Individual Phage Research Articles

BACPHLIP: predicting bacteriophage lifestyle from conserved protein domains.

Bacteriophages are broadly classified into two distinct lifestyles: temperate and virulent. Temperate phages are capable of a latent phase of infection within a host cell (lysogenic cycle), whereas virulent phages directly replicate and lyse host cells upon infection (lytic cycle). Accurate lifestyle identification is critical for determining the role of individual phage species within ecosystems and their effect on host evolution. Here, we present BACPHLIP, a BACterioPHage LIfestyle Predictor. BACPHLIP detects the presence of a set of conserved protein domains within an input genome and uses this data to predict lifestyle via a Random Forest classifier that was trained on a dataset of 634 phage genomes. On an independent test set of 423 phages, BACPHLIP has an accuracy of 98% greatly exceeding that of the previously existing tools (79%). BACPHLIP is freely available on GitHub (https://github.com/adamhockenberry/bacphlip) and the code used to build and test the classifier is provided in a separate repository (https://github.com/adamhockenberry/bacphlip-model-dev) for users wishing to interrogate and re-train the underlying classification model.

Dietary Modulation of Bacteriophages as an Additional Player in Inflammation and Cancer.

Simple SummaryThe role and function of bacteriophages (phages) in the intestine, its health and microbial homeostasis has been underestimated so far. This interdisciplinary review highlights the effect of dietary compounds on phages and puts this into perspective with putative contributions of phages to gastrointestinal diseases, specifically inflammation, infection, and cancer. The review discusses novel fields of opportunities in this context. These include, but are not limited to, perspectives how a better understanding of modulating the activity of specific phages by particular nutritional components may contribute to reorganizing the microbial network, thus supporting in the combat, or even prevention, of inflammation or even cancer in the gut.Natural compounds such as essential oils and tea have been used successfully in naturopathy and folk medicine for hundreds of years. Current research is unveiling the molecular role of their antibacterial, anti-inflammatory, and anticancer properties. Nevertheless, the effect of these compounds on bacteriophages is still poorly understood. The application of bacteriophages against bacteria has gained a particular interest in recent years due to, e.g., the constant rise of antimicrobial resistance to antibiotics, or an increasing awareness of different types of microbiota and their potential contribution to gastrointestinal diseases, including inflammatory and malignant conditions. Thus, a better knowledge of how dietary products can affect bacteriophages and, in turn, the whole gut microbiome can help maintain healthy homeostasis, reducing the risk of developing diseases such as diverse types of gastroenteritis, inflammatory bowel disease, or even cancer. The present review summarizes the effect of dietary compounds on the physiology of bacteriophages. In a majority of works, the substance class of polyphenols showed a particular activity against bacteriophages, and the primary mechanism of action involved structural damage of the capsid, inhibiting bacteriophage activity and infectivity. Some further dietary compounds such as caffeine, salt or oregano have been shown to induce or suppress prophages, whereas others, such as the natural sweeter stevia, promoted species-specific phage responses. A better understanding of how dietary compounds could selectively, and specifically, modulate the activity of individual phages opens the possibility to reorganize the microbial network as an additional strategy to support in the combat, or in prevention, of gastrointestinal diseases, including inflammation and cancer.

Bacteriophage therapy for challenging bacterial infections: achievements, limitations and prospects for future clinical use by veterinary dermatologists.

Bacteriophages were discovered just over 100 years ago and have been used to treat bacterial infections in animals since the 1920s. The antimicrobial resistance crisis has led to a new surge of interest in the use of bacteriophage therapy as an alternative or supplement to antimicrobial therapy in humans and other animals. To describe the nature of bacteriophages and provide a critical review and update on the clinical use of bacteriophages in the treatment of challenging bacterial infections, with an emphasis on companion animal veterinary applications. The scientific literature on the subject was critically evaluated. Findings from the most pertinent papers have been presented in summary form and critiqued. Over the last 20 years there has been a considerable increase in the volume and quality of publications dealing with bacteriophage therapy. Some recent papers build on excellent work published in the 1980s and describe promising veterinary applications. Challenges related particularly to the registration and approval of phage remedies will need to be overcome before phage therapy can become a mainstream tool for use in veterinary settings. Considerably more research, particularly controlled clinical trials, needs to be done. Bacteriophage therapy is one of the most promising approaches to tackling the looming antimicrobial resistance crisis, yet substantial regulatory challenges will need to be overcome before it enters widespread use. Phage therapy also may, in the future, improve the management of challenging bacterial infections that are not necessarily multidrug-resistant.

Primate phageomes are structured by superhost phylogeny and environment

Humans harbor diverse communities of microorganisms, the majority of which are bacteria in the gastrointestinal tract. These gut bacterial communities in turn host diverse bacteriophage (hereafter phage) communities that have a major impact on their structure, function, and, ultimately, human health. However, the evolutionary and ecological origins of these human-associated phage communities are poorly understood. To address this question, we examined fecal phageomes of 23 wild nonhuman primate taxa, including multiple representatives of all the major primate radiations. We find relatives of the majority of human-associated phages in wild primates. Primate taxa have distinct phageome compositions that exhibit a clear phylosymbiotic signal, and phage-superhost codivergence is often detected for individual phages. Within species, neighboring social groups harbor compositionally and evolutionarily distinct phageomes, which are structured by superhost social behavior. Captive nonhuman primate phageome composition is intermediate between that of their wild counterparts and humans. Phage phylogenies reveal replacement of wild great ape-associated phages with human-associated ones in captivity and, surprisingly, show no signal for the persistence of wild-associated phages in captivity. Together, our results suggest that potentially labile primate-phage associations have persisted across millions of years of evolution. Across primates, these phylosymbiotic and sometimes codiverging phage communities are shaped by transmission between groupmates through grooming and are dramatically modified when primates are moved into captivity.

Isolation and Characterization of Klebsiella Phages for Phage Therapy.

Introduction: Klebsiella is a clinically important pathogen causing a variety of antimicrobial resistant infections in both community and nosocomial settings, particularly pneumonia, urinary tract infection, and sepsis. Bacteriophage (phage) therapy is being considered a primary option for the treatment of drug-resistant infections of these types.Methods: We report the successful isolation and characterization of 30 novel, genetically diverse Klebsiella phages.Results: The isolated phages span six different phage families and nine genera, representing both lysogenic and lytic lifestyles. Individual Klebsiella phage isolates infected up to 11 of the 18 Klebsiella capsule types tested, and all 18 capsule-types were infected by at least one of the phages.Conclusions: Of the Klebsiella-infecting phages presented in this study, the lytic phages are most suitable for phage therapy, based on their broad host range, high virulence, short lysis period and given that they encode no known toxin or antimicrobial resistance genes. Phage isolates belonging to the Sugarlandvirus and Slopekvirus genera were deemed most suitable for phage therapy based on our characterization. Importantly, when applied alone, none of the characterized phages were able to suppress the growth of Klebsiella for more than 12 h, likely due to the inherent ease of Klebsiella to generate spontaneous phage-resistant mutants. This indicates that for successful phage therapy, a cocktail of multiple phages would be necessary to treat Klebsiella infections.

Efficacy of Individual Bacteriophages Does Not Predict Efficacy of Bacteriophage Cocktails for Control of Escherichia coli O157.

Effectiveness of bacteriophages AKFV33 (Tequintavirus, T5) and AHP24 (Rogunavirus, T1), wV7 (Tequatrovirus, T4), and AHP24S (Vequintavirus, rV5), as well as 11 cocktails of combinations of the four phages, were evaluated in vitro for biocontrol of six common phage types of Escherichia coli O157 (human and bovine origins) at different multiplicities of infection (MOIs; 0.01–1,000), temperatures (37 or 22°C), and exposure times (10–22 h). Phage efficacy against O157 was highest at MOI 1,000 (P < 0.001) and after 14-18 h of exposure at 22°C (P < 0.001). The activity of individual phages against O157 did not predict the activity of a cocktail of these phages even at the same temperature and MOI. Combinations of phages were neutral (no better or worse than the most effective constituent phages acting alone), displayed facilitation (greater efficacy than the most effective constituent phages acting alone), or antagonistic (lower efficacy than the most effective constituent phages acting alone). Across MOIs, temperatures, exposure time, and O157 strains, a cocktail of T1, T4, and rV5 was most effective (P < 0.05) against O157, although T1 and rV5 were less effective (P < 0.001) than other individual phages. T5 was the most effective individual phages (P < 0.05), but was antagonistic to other phages, particularly rV5 and T4 + rV5. Interactions among phages were influenced by phage genera and phage combination, O157 strains, MOIs, incubation temperatures, and times. Based on this study, future development of phage cocktails should, as a minimum, include confirmation of a lack of antagonism among constituent phages and preferably confirmation of facilitation or synergistic effects.

An Optimized Bacteriophage Cocktail Can Effectively Control Salmonella in vitro and in Galleria mellonella.

Salmonella spp. is a leading cause of gastrointestinal enteritis in humans where it is largely contracted via contaminated poultry and pork. Phages can be used to control Salmonella infection in the animals, which could break the cycle of infection before the products are accessible for consumption. Here, the potential of 21 myoviruses and a siphovirus to eliminate Salmonella in vitro and in vivo was examined with the aim of developing a biocontrol strategy to curtail the infection in poultry and swine. Together, the phages targeted the twenty-three poultry and ten swine prevalent Salmonella serotype isolates tested. Although individual phages significantly reduced bacterial growth of representative isolates within 6 h post-infection, bacterial regrowth occurred 1 h later, indicating proliferation of resistant strains. To curtail bacteriophage resistance, a novel three-phage cocktail was developed in vitro, and further investigated in an optimized Galleria mellonella larva Salmonella infection model colonized with representative swine, chicken and laboratory strains. For all the strains examined, G. mellonella larvae given phages 2 h prior to bacterial exposure (prophylactic regimen) survived and Salmonella was undetectable 24 h post-phage treatment and throughout the experimental time (72 h). Administering phages with bacteria (co-infection), or 2 h post-bacterial exposure (remedial regimen) also improved survival (73–100% and 15–88%, respectively), but was less effective than prophylaxis application. These pre-livestock data support the future application of this cocktail for further development to effectively treat Salmonella infection in poultry and pigs. Future work will focus on cocktail formulation to ensure stability and incorporation into feeds and used to treat the infection in target animals.

In vivo phage display: identification of organ-specific peptides using deep sequencing and differential profiling across tissues.

In vivo phage display is widely used for identification of organ- or disease-specific homing peptides. However, the current in vivo phage biopanning approaches fail to assess biodistribution of specific peptide phages across tissues during the screen, thus necessitating laborious and time-consuming post-screening validation studies on individual peptide phages. Here, we adopted bioinformatics tools used for RNA sequencing for analysis of high-throughput sequencing (HTS) data to estimate the representation of individual peptides during biopanning in vivo. The data from in vivo phage screen were analyzed using differential binding—relative representation of each peptide in the target organ versus in a panel of control organs. Application of this approach in a model study using low-diversity peptide T7 phage library with spiked-in brain homing phage demonstrated brain-specific differential binding of brain homing phage and resulted in identification of novel lung- and brain-specific homing peptides. Our study provides a broadly applicable approach to streamline in vivo peptide phage biopanning and to increase its reproducibility and success rate.

Host diversity slows bacteriophage adaptation by selecting generalists over specialists.

Most viruses can infect multiple hosts, yet the selective mechanisms that maintain multi-host generalists over single-host specialists remain an open question. Here we propagate populations of the newly identified bacteriophage øJB01 in coculture with many host genotypes and find that while phage can adapt to infect any of the new hosts, increasing the number of hosts slows the rate of adaptation. We quantify trade-offs in the capacity for individual phage to infect different hosts and find that phage from evolved populations with more hosts are more likely to be generalists. Sequencing of evolved phage reveals strong selection and the genetic basis of adaptation, supporting a model that shows how the addition of more potential hosts to a community can select for low-fitness generalists over high-fitness specialists. Our results show how evolution with multiple hosts alters the rate of viral adaptation and provides empirical support for an evolutionary mechanism that promotes generalists over specialists.

Characterization of four virulent Klebsiella pneumoniae bacteriophages, and evaluation of their potential use in complex phage preparation

BackgroundNowadays, hundreds of thousands of deaths per year are caused by antibiotic resistant nosocomial infections and the prognosis for future years is much worse, as evidenced by modern research. Bacteria of the Klebsiella genus are one of the main pathogens that cause nosocomial infections. Among the many antimicrobials offered to replace or supplement traditional antibiotics, bacteriophages are promising candidates.MethodsThis article presents microbiological, physicochemical and genomic characterization of 4 virulent bacteriophages belonging to Siphoviridae, Myoviridae and Podoviridae families. Phages were studied by electron microscopy; their host range, lytic activity, adsorption rate, burst size, latent period, frequency of phage-resistant forms generation, lysis dynamics and sensitivity of phage particles to temperature and pH were identified; genomes of all 4 bacteriophages were studied by restriction digestion and complete genome sequence.ResultsStudied phages showed wide host range and high stability at different temperature and pH values. In contrast with single phages, a cocktail of bacteriophages lysed all studied bacterial strains, moreover, no cases of the emergence of phage-resistant bacterial colonies were detected. Genomic data proved that isolated viruses do not carry antibiotic resistance, virulence or lysogenic genes. Three out of four bacteriophages encode polysaccharide depolymerases, which are involved in the degradation of biofilms and capsules.ConclusionsThe bacteriophages studied in this work are promising for further in vivo studies and might be used in phage therapy as part of a complex therapeutic and prophylactic phage preparation. The conducted studies showed that the complex preparation is more effective than individual phages. The use of the complex phage cocktail allows to extend the lytic spectrum, and significantly reduces the possibility of phage-resistant forms generation.

Lysogenization of a Lactococcal Host with Three Distinct Temperate Phages Provides Homologous and Heterologous Phage Resistance.

Lactococcus lactis is the most widely exploited microorganism in global dairy fermentations. Lactococcal strains are described as typically harboring a number of prophages in their chromosomes. The presence of such prophages may provide both advantages and disadvantages to the carrying host. Here, we describe the deliberate generation of three distinct lysogens of the model lactococcal strain 3107 and the impact of additional prophage carriage on phage-resistance and anti-microbial susceptibility. Lysogen-specific responses were observed, highlighting the unique relationship and impact of each lysogenic phage on its host. Both homologous and heterologous phage-resistance profiles were observed, highlighting the presence of possible prophage-encoded phage-resistance factors. Superinfection exclusion was among the most notable causes of heterologous phage-resistance profiles with resistance observed against members of the Skunavirus, P335, P087, and 949 lactococcal phage groups. Through these analyses, it is now possible to identify phages that may pursue similar DNA injection pathways. The generated lysogenic strains exhibited increased sensitivity to the antimicrobial compounds, nisin and lysozyme, relative to the parent strain, although it is noteworthy that the degree of sensitivity was specific to the individual (pro)phages. Overall, the findings highlight the unique impact of each prophage on a given strain and the requirement for strain-level analysis when considering the implications of lysogeny.

Contribution of Podoviridae and Myoviridae bacteriophages to the effectiveness of anti-staphylococcal therapeutic cocktails

Bacteriophage therapy is considered one of the most promising therapeutic approaches against multi-drug resistant bacterial infections. Infections caused by Staphylococcus aureus are very efficiently controlled with therapeutic bacteriophage cocktails, containing a number of individual phages infecting a majority of known pathogenic S. aureus strains. We assessed the contribution of individual bacteriophages comprising a therapeutic bacteriophage cocktail against S. aureus in order to optimize its composition. Two lytic bacteriophages vB_SauM-515A1 (Myoviridae) and vB_SauP-436A (Podoviridae) were isolated from the commercial therapeutic cocktail produced by Microgen (Russia). Host ranges of the phages were established on the panel of 75 S. aureus strains. Phage vB_SauM-515A1 lysed 85.3% and vB_SauP-436A lysed 68.0% of the strains, however, vB_SauP-436A was active against four strains resistant to vB_SauM-515A1, as well as to the therapeutic cocktail per se. Suboptimal results of the therapeutic cocktail application were due to extremely low vB_SauP-436A1 content in this composition. Optimization of the phage titers led to an increase in overall cocktail efficiency. Thus, one of the effective ways to optimize the phage cocktails design was demonstrated and realized by using bacteriophages of different families and lytic spectra.

Efficacy of novel phages for control of multi-drug resistant Escherichia coli O177 on artificially contaminated beef and their potential to disrupt biofilm formation

Contaminated beef is a prominent source of foodborne pathogens such as Escherichia coli O177. Susceptibility of nine multi-drug resistant E. coli O177 strains against eight individual phages and six phage cocktails was assessed using polystyrene microplate titer plate. Further, 180 beef samples were independently inoculated with E. coli O177 cells in triplicates and treated with eight individual phages and six phage cocktails to determine their efficacy in inhibiting bacteria growth at 4 °C over a 7-day incubation period. Results revealed that all E. coli O177 strains were susceptible to the phages. A significant log reduction in viable E. coli O177 cell counts was observed on beef samples upon phage treatment over the 7-day incubation period. Two individual phages and three phage cocktails reduced E. coli cell counts to levels below the detection limit (1.0 log10 CFU/g). Log reduction of viable E. coli cell counts ranged from 2.10 to 7.81 CFU/g for individual phages and from 2.86 to 7.81 CFU/g for cocktails. Individual phages and phage cocktails inhibited E. coli O177 biofilm formation with phage cocktails showing high efficacy. Furthermore, phage cocktails showed greater efficacy in destroying pre-formed biofilm than individual phages. Based on these findings, we concluded that phage cocktails developed in this study could be used to reduce E. coli O177 contamination and extend the shelf-life of stored raw beef.

Lysis Performance of Bacteriophages with Different Plaque Sizes and Comparison of Lysis Kinetics After Simultaneous and Sequential Phage Addition.

Background: Although bacteriophages see a revival for specifically removing undesired bacteria, there is still much uncertainty about how to achieve the most rapid and long-lasting clearance. Materials and Methods: This study investigated the lysis kinetics of three distinct environmental coliphages, reproducibly forming different plaque sizes (big, medium, and small). Lysis performance by individual phages was compared with the one obtained after simultaneous or sequential addition of all three phages. Kinetics was monitored by density absorbance or by flow cytometry, with the latter having the advantage of providing higher sensitivity. Results: Plaque size happened to correlate with lysis kinetics in liquid suspensions, with phages producing big (phage B), medium (phage M), and small (phage S) plaques showing maximal bacterial clearance under the chosen conditions within ∼6, 12, and 18 h, respectively. Use of a phage cocktail (all three phages added simultaneously) resulted in slower initial lysis compared with the fastest lysing phage with the greatest plaque size alone, but it showed longer efficacy in suppression. When adding phages sequentially, overall lysis kinetics could be influenced by administering phages at different time points. The lowest bacterial concentration after 36 h was obtained when administering phages in the sequence S, M, and B although this combination initially took the longest to achieve bacterial clearance. Conclusions: Results support that timing and order of phage addition can modulate strength and duration of bacterial suppression and, thus, influence the overall success of phage treatment.

Emerging heterogeneous compartments by viruses in single bacterial cells

Spatial organization of biological processes allows for variability in molecular outcomes and coordinated development. Here, we investigate how organization underpins phage lambda development and decision-making by characterizing viral components and processes in subcellular space. We use live-cell and in situ fluorescence imaging at the single-molecule level to examine lambda DNA replication, transcription, virion assembly, and resource recruitment in single-cell infections, uniting key processes of the infection cycle into a coherent model of phage development encompassing space and time. We find that different viral DNAs establish separate subcellular compartments within cells, which sustains heterogeneous viral development in single cells. These individual phage compartments are physically separated by the E. coli nucleoid. Our results provide mechanistic details describing how separate viruses develop heterogeneously to resemble single-cell phenotypes.

Efficacy of commercial bacteriophage products against ESKAPE pathogens

The ever-rising prevalence of multidrug-resistant bacteria necessitates the search for a therapeutic alternative to antibiotics. Using therapeutic products based on virulent bacteriophages might provide such an alternative. The aim of our study was to evaluate the efficacy of commercial phage products and natural bacteriophage monoisolates recovered from environmental sources against clinical strains of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. We compiled a collection of 147 strains that were subsequently genotypes using the MLST method. The efficacy of bacteriophages was evaluated in spot tests. The highest efficacy was demonstrated by "Staphylococcal bacteriophage" (86%, effective against S. aureus), "Purified polyvalent pyobacteriophage" (87.8%, effective against K. pneumoniae), and a group of phage products against P. aeruginosa, including "Pseudomonas aeruginosa bacteriophage" (87.5%), "Complex pyobacteriophage" (79.5–90%) and "Purified polyvalent pyobacteriophage" (90–92.5%). The efficacy of "Intesti bacteriophage", which targets E. faecium, was 4.2%. The efficacy of commercial phage products against S. aureus and K. pneumoniae was higher than the efficacy of individual phage monoisolates (60% for the S. aureus phage vB_SauP-436-3w and 5.9% for the K. pneumoniae phage vB_Kp_M_ Seu621). Thus, all tested commercial phage products were highly effective against P. aeruginosa, K. pneumoniae and S. aureus. There are no commercial phage products on the market against other ESKAPE pathogens, including Acinetobacter baumannii and Enterobacter cloacae. Besides, there are no effective phage products against E. faecium. This dictates the need for new effective bacteriophages against these species.

InteractomeSeq: a web server for the identification and profiling of domains and epitopes from phage display and next generation sequencing data.

High-Throughput Sequencing technologies are transforming many research fields, including the analysis of phage display libraries. The phage display technology coupled with deep sequencing was introduced more than a decade ago and holds the potential to circumvent the traditional laborious picking and testing of individual phage rescued clones. However, from a bioinformatics point of view, the analysis of this kind of data was always performed by adapting tools designed for other purposes, thus not considering the noise background typical of the ‘interactome sequencing’ approach and the heterogeneity of the data. InteractomeSeq is a web server allowing data analysis of protein domains (‘domainome’) or epitopes (‘epitome’) from either Eukaryotic or Prokaryotic genomic phage libraries generated and selected by following an Interactome sequencing approach. InteractomeSeq allows users to upload raw sequencing data and to obtain an accurate characterization of domainome/epitome profiles after setting the parameters required to tune the analysis. The release of this tool is relevant for the scientific and clinical community, because InteractomeSeq will fill an existing gap in the field of large-scale biomarkers profiling, reverse vaccinology, and structural/functional studies, thus contributing essential information for gene annotation or antigen identification. InteractomeSeq is freely available at https://InteractomeSeq.ba.itb.cnr.it/

Bacteriophages as promising agents for the biological control of Moko disease (Ralstonia solanacearum) of banana

“Moko disease”, caused by the bacterium Ralstonia solanacearum, is one of the most devastating diseases of banana and plantain. Its management is difficult, given its aggressiveness and easy dispersion, as well as the lack of products for its control. Effectiveness of antibiotics is quite low and their use in the field is restricticted, given environmental and human health concerns. Bacteriophages (phages) have emerged as an alternative for the control of bacterial plant diseases, but their potential for the control of Moko is yet to be tested. Thus, in this study, phages isolated from soils cultivated with banana and plantain were selected and evaluated for their capacity to control R. solanacearum, both in vitro and on banana plants under greenhouse conditions. For this, eight lytic phages were isolated from soil collected in plantations of banana and plantain in Colombia and tested against 65 native strains of R. solanacearum. The two most effective phages were further evaluated for their capacity to inhibit the growth in vitro and in a soil microcosm of a highly pathogenic strain, R. solanacearum UA1591. Here, both phages reduced the growth of the pathogen in liquid media and soil microcosm to levels that were below the threshold of detection in 24 h. Lastly, in a greenhouse experiment, plants treated with the two-phage cocktail were 100% protected from Moko, even though most plants treated with individual phages still displayed symptoms of the disease. These results support that two-phage cocktails are promising biological control agents against Moko disease.

Nano-etched fused-silica capillary used for on-line preconcentration and electrophoretic separation of bacteriophages from large blood sample volumes with off-line MALDI-TOF mass spectrometry identification.

The properties of staphylococcal phages from the Siphoviridae, Podoviridae, and Myoviridae families were monitored using capillary electrophoretic methods on fused-silica capillaries with different morphology of surface roughness. Isoelectric points of the examined phages were determined by capillary isoelectric focusing in the original, smooth fused-silica capillary, and they ranged from 3.30 to 3.85. For capillary electrophoresis of phages, fused-silica capillaries with the "pock" and "cone" roughened surface types were prepared by etching a part of the capillary with supercritical water. The best resolution of the individual phages (to range from 3.2 to 4.6) was achieved with the "cone" surface-type fused-silica capillary. Direct application of phage K1/420 at the infection site, represented by human plasma or full blood spiked with Staphylococcus aureus, was on-line monitored by micellar electrokinetic chromatography. The phage particles were dynamically adhered onto the roughened surface of the capillary from 10μL of the prepared sample at the optimized flow rate of 6.5μLmin-1. The limit of detection was determined to be 104 phage particles. The linearity of the calibration lines was characterized by the regression coefficient, R2 =0.998. The relative standard deviation (RSD) of the peak area, calculated from ten independent measurements, was (±) 2%. After analysis, viability of the detected phages was verified by the modified "double-layer drop assay" method, and collected phage fractions were simultaneously off-line analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Graphical abstract.

Effects of Bacteriophage, Ultraviolet Light, and Organic Acid Applications on STEC O157:H7 and the “Big Six” in Beef Packaged Under Vacuum and Aerobic Conditions

ObjectivesBeef primals produced during high event periods (HEP) can also be affected by STEC contamination requiring microbial assessment. Commonly, primals are retreated with antimicrobials after removal from vacuum bags, then repackaged and tested for STEC. In this study, we evaluated the efficiency of bacteriophage, ultraviolet light, and organic acids on contaminated beef kept under vacuum and aerobic conditions.Materials and MethodsThe effects of antimicrobial interventions Peroxyacetic acid (PAA, 400 PPM), Ultraviolet light (UV, 30 s at 2.5 ± 0.3 cm height), Acidified Sodium Chlorite (ASC, 1200 ppm), and bacteriophage (P, 7 MS phages at 108 PFU/ml) against STEC (O157:H7 and O145, O121, O111, O103, O45, O26) were evaluated on beef. Fresh m. cutaneous trunci was fabricated into 100 cm2 samples (n = 154), which were randomly assigned to 11 treatments including Control, P, UV, ASC, PAA, and combinations P+UV, P+PAA, P+ASC, UV+PAA, UV+ASC, PAA+ASC. Treatments were tested under vacuum and aerobic conditions. Samples were inoculated with a STEC cocktail comprised of 7 strains to yield 3 log CFU/cm2. Samples were vacuumed or overwrapped with oxygen permeable film. Samples were unpackaged and treated with buffered peptone water (BPW, Control) or individual or combined antimicrobial treatments prior to re-packaging. After 1 h at 7°C, samples were swabbed, homogenized in 1 mL of BPW, serially diluted and spread-plated for bacterial enumeration. Data was analyzed using SAS as a completely randomized design.ResultsOverall, treatments including MS phages significantly decreased STEC populations in beef under vacuum and aerobic conditions (P &lt; 0.0001). Under vacuum, individual phage application, combinations between phage and UV, ASC, and PAA plus UV+ASC provided optimal STEC reduction on beef surface. Phage and PAA combination led to the lowest STEC load (1.49 log reduction). When analyzing contrasts, treatments with phage significantly decreased STEC loads when compared to other treatments (P &lt; 0.0001) and control (P &lt; 0.0001). STEC loads recovered from treatments without phage and control were statistically similar at P = 0.32. Under aerobic conditions, individual treatments UV and ASC and combinations including UV+PAA, and PAA+ASC were statistically similar to the control. Inclusion of phage in treatments gradually decreased STEC loads when combined with ASC, PAA, and UV. Phage and UV combination led to the lowest STEC load (1.46 log reduction). Contrast analysis showed that treatments with phage significantly decreased STEC loads when compared to other treatments (P &lt; 0.0001) and control (P &lt; 0.0001). STEC loads recovered from treatments without phage and control were statistically similar at P = 0.07.ConclusionIndividual or combined applications of MS phages on beef surface contaminated with STEC provided optimal antimicrobial effect under vacuum or aerobic conditions. Although organic acids and UV combinations did reduce STEC populations, treatments that included phage yielded the lowest STEC loads. Only phage interventions gave optimal reduction effects under vacuum conditions. Antimicrobial treatments based on individual phage cocktails and their combinations with ASC, UV, and PAA significantly reduce STEC when treating primals produced during HEP.