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Antimicrobial resistance is increasing despite new treatments being employed. With a decrease in the discovery rate of novel antibiotics, this threatens to take humankind back to a “pre-antibiotic era” of clinical care. Bacteriophages (phages) are one of the most promising alternatives to antibiotics for clinical use. Although more than a century of mostly ad-hoc phage therapy has involved substantial clinical experimentation, a lack of both regulatory guidance standards and effective execution of clinical trials has meant that therapy for infectious bacterial diseases has yet to be widely adopted. However, several recent case studies and clinical trials show promise in addressing these concerns. With the antibiotic resistance crisis and urgent search for alternative clinical treatments for bacterial infections, phage therapy may soon fulfill its long-held promise. This review reports on the applications of phage therapy for various infectious diseases, phage pharmacology, immunological responses to phages, legal concerns, and the potential benefits and disadvantages of this novel treatment.

There is growing concern about the emergence of bacterial strains showing resistance to all classes of antibiotics commonly used in human medicine. Despite the broad range of available antibiotics, bacterial resistance has been identified for every antimicrobial drug developed to date. Alarmingly, there is also an increasing prevalence of multidrug-resistant bacterial strains, rendering some patients effectively untreatable. Therefore, there is an urgent need to develop alternatives to conventional antibiotics for use in the treatment of both humans and food-producing animals. Bacteriophage-encoded lytic enzymes (endolysins), which degrade the cell wall of the bacterial host to release progeny virions, are potential alternatives to antibiotics. Preliminary studies show that endolysins can disrupt the cell wall when applied exogenously, though this has so far proven more effective in Gram-positive bacteria compared with Gram-negative bacteria. Their potential for development is furthered by the prospect of bioengineering, and aided by the modular domain structure of many endolysins, which separates the binding and catalytic activities into distinct subunits. These subunits can be rearranged to create novel, chimeric enzymes with optimized functionality. Furthermore, there is evidence that the development of resistance to these enzymes may be more difficult compared with conventional antibiotics due to their targeting of highly conserved bonds.

Livestock-associated Staphylococcus aureus ST398 is a highly pathogenic species that causes infections in a wide variety of animals, including humans. The bacteriophage (phage) vB_SauS_S1 was isolated originally using a ST398 strain as its “isolating host”, then the spot tests showed it was able to infect 73.33% (22/30) ST398 isolates. Phage S1 was assigned as a temperate phage based on genome analysis and phenotypic validation. Phylogenetic analysis showed that S1 was closely related to temperate phages tp310-2 and SA137ruMSSAST121PVL. Following infection of ST398 by phage S1, the lysogenic strain showed enhanced biofilm forming ability compared to the wildtype strain, and the invasion rate of MAC-T cells increased by 10.39%. The minimum inhibitory concentration showed that phage S1 did not change the antibiotic sensitivity of the lysogen strain, and the virulence of the lysogen strain did not change significantly in the injection models of Galleria mellonella ( G. mellonella ) and mice. The lysogen demonstrated superinfection immunity and reduced sensitivity to virulent phage infection. Thus, this study contributes to understanding the co-evolutionary relationships between temperate phages and the multi-host zoonotic pathogen S. aureus ST398.

Livestock-associated Staphylococcus aureus ST398 is a highly pathogenic species that causes infections in a wide variety of animals, including humans. The bacteriophage (phage) vB_(S)auS_(S)1 was isolated originally using a ST398 strain as its \"isolating host\", then the spot tests showed it was able to infect 73.33% (22/30) ST398 isolates. Phage S1 was assigned as a temperate phage based on genome analysis and phenotypic validation. Phylogenetic analysis showed that S1 was closely related to temperate phages tp310-2 and SA137ruMSSAST121PVL. Following infection of ST398 by phage S1, the lysogenic strain showed enhanced biofilm forming ability compared to the wildtype strain, and the invasion rate of MAC-T cells increased by 10.39%. The minimum inhibitory concentration showed that phage S1 did not change the antibiotic sensitivity of the lysogen strain, and the virulence of the lysogen strain did not change significantly in the injection models of Galleria mellonella (G. mellonella) and mice. The lysogen demonstrated superinfection immunity and reduced sensitivity to virulent phage infection. Thus, this study contributes to understanding the co-evolutionary relationships between temperate phages and the multi-host zoonotic pathogen S. aureus ST398.

Antimicrobial resistance threatens a new “dark age” in medical practice. Chronic antibiotic overuse has driven the rise in antimicrobial resistance and promoted the emergence of multidrug-resistant organisms. To address this problem, researchers have developed new approaches. Antimicrobials derived from bacteriophage, which are viruses that target bacteria, are promising candidates. Amongst these candidates, bacteriophage enzymes used in the viral replication cycle are of significant interest. Specifically, endolysins are used by bacteriophage to lyse the bacterial cell wall, leading to structural collapse and cell lysis. Researchers are increasingly applying these proteins externally to multidrug-resistant organisms as a novel antimicrobial treatment. Following this increased interest, many studies have presented protein engineering methods to further enhance the effectiveness of endolysins as antimicrobials. These methods include attachment of membrane-permeabilizing peptides, domain-swapping, and catalytic-site modification. Recent advances in all three fields have seen the implementation of tools like novel in silico design pipelines and library-based screening methods. This review summarizes these recent advances in the rapidly developing field of endolysin engineering and discusses potential future directions in this field.

Bacteriophage-encoded endolysins have been identified as antibacterial candidates. However, the development of endolysins as mainstream antibacterial agents first requires a comprehensive biochemical understanding. This study defines the atomic structure and enzymatic function of Escherichia coli O157:H7 phage FAHEc1 endolysin, LysF1. Bioinformatic analysis suggests this endolysin belongs to the T4 Lysozyme (T4L)-like family of proteins and contains a highly conserved catalytic triad. We then solved the structure of LysF1 with x-ray crystallography to 1.71 Å. LysF1 was confirmed to exist as a monomer in solution by sedimentation velocity experiments. The protein architecture of LysF1 is conserved between T4L and related endolysins. Comparative analysis with related endolysins shows that the spatial orientation of the catalytic triad is conserved, suggesting the catalytic mechanism of peptidoglycan degradation is the same as that of T4L. Differences in the sequence illustrate the role coevolution may have in the evolution of this fold. We also demonstrate that by mutating a single residue within the hydrophobic core, the thermal stability of LysF1 can be increased by 9.4 °C without compromising enzymatic activity. Overall, the characterization of LysF1 provides further insight into the T4L-like class of endolysins. Our study will help advance the development of related endolysins as antibacterial agents, as rational engineering will rely on understanding mutable positions within this protein fold.

Food-borne diseases are a global public health issue with 1 in 10 people falling ill after eating contaminated food every year. In response, the food industry has implemented several new pathogen control strategies, such as biotechnological tools using the direct application of bacteriophages for biological control. We have undertaken a systematic review and meta-analysis that evaluated the efficiency of patented phages as a biological control for food-borne pathogens and determined the physical–chemical characteristics of the antimicrobial effect. Included and excluded criteria was developed. Included criteria: Phage patent files with an application in biological control on food and scientific articles and book chapters that used phages patented for food biological control. Excluded criteria: P atent documents, scientific articles, and book chapters that included phage therapy in humans, animals, and biological control on plants but did not have an application on food were not considered in our study. The systematic analysis identified 77 documents, 46 scientific articles, and 31 documents of patents and 23 articles was included in the meta-analysis. Listeria monocytogenes and Salmonella sp. comprised most of the targets identified in the screening, so that we focused on these strains to do the meta-analysis. There are a total of 383 and 192 experiments for Listeria and Salmonella phages for quantitative data analysis. Indexing databases for the bibliographic search (Scopus, Web of Science (WoS) and PubMed (Medline) were addressed by an automated script written in Python 3 Python Core Team (2015) and deposited on GitHub ( https://github.com/glenjasper ). A random-effects meta-analysis revealed (i) significant antimicrobial effect of Listeria phages in apple, apple juice, pear, and pear juice, (ii) significant antimicrobial effect of Salmonella phages in eggs, apple, and ready-to-eat chicken, (iii) no heterogeneity was identified in either meta-analysis, (iv) publication bias was detected for Listeria phages but not for Salmonella phages. (v) ListShield and Felix01 phages showed the best result for Listeria and Salmonella biological control, respectively, (vi) concentration of phage and bacteria, time and food had significant effect in the biological control of Listeria , (vii) temperature and time had a significant effect on the antimicrobial activity of Salmonella phages. The systematic review and meta-analyses to determine the efficiency of bacteriophages previously patented against pathogenic bacteria on dairy products, meat, fruits and vegetables. Besides, the discovering of key factors for efficacy, so that future applications of phage biotechnology in foods can be optimally deployed.

Cronobacter spp. are the most concerning foodborne pathogen in infant formula milk powder. Currently, there are many reports on the prevalence of Cronobacter spp. in infant formula milk and its processing environment, but there are few studies on the prevalence of Cronobacter spp. on dairy farms. We have, therefore, undertaken this study to investigate and track genomic epidemiology of Cronobacter spp. isolates from Chinese dairy farms in the provinces of Jiangsu and Shandong. In this study, forty Cronobacter spp. strains, consisting of thirty Cronobacter sakazakii, eight Cronobacter malonaticus, and two Cronobacter dublinensis, were obtained from 1115 dairy farm samples (raw milk, silage, bedding, and feces), with a prevalence rate of 3.57%. These isolates were classified into 10 Cronobacter serotypes and 31 sequence types (STs), including three novel STs which were isolated for the first time. Notably, pathogenic Cronobacter STs 7, 8, 17, 60, and 64, which are associated with clinical infections, were observed. Antimicrobial susceptibility testing showed that all the Cronobacter spp. were highly resistant to cephalothin and fosfomycin, which was consistent with the antimicrobial genotype. All isolates carried core virulence genes related to adherence, invasion, endotoxin, immune evasion, secretion system, and regulation. Approximately half the isolates were also able to produce a strong biofilm. Twenty-one prophages and eight plasmids were detected, with the most common prophage being Cronobacter_ENT47670 and the most common plasmid being IncFIB (pCTU1). In addition, two isolates harbored the transmissible locus of stress tolerance (tLST) which confers high environmental persistence. Phylogenetic analysis showed strong clustering by species level and sequence types. Isolates from different sources or regions with a similar genomic background suggests the cross-contamination of Cronobacter spp. The presence of diverse genotypes of Cronobacter spp. in dairy farms in Jiangsu and Shandong provinces indicates that surveillance of Cronobacter spp. on dairy farms should be strengthened, to prevent and control transmission and ensure the quality and safety of raw dairy products.

Free-living amoebae (FLA) are common in both natural and engineered freshwater ecosystems. They play important roles in biofilm control and contaminant removal through the predation of bacteria and other taxa. Bacterial predation by FLA is also thought to contribute to pathogen dispersal and infectious disease transmission in freshwater environments via the egestion of viable bacteria. Despite their importance in shaping freshwater microbial communities, the diversity and function of FLA in many freshwater ecosystems are poorly understood. In this study, we isolated and characterized FLA from two groundwater sites in Canterbury, New Zealand using microbiological, microscopic, and molecular techniques. Different methods for groundwater FLA isolation and enrichment were trialed and optimized. The ability of these isolated FLA to predate on human pathogen Legionella pneumophila was assessed. FLA were identified by 18S metagenomic amplicon sequencing. Our study showed that Acanthamoeba spp. (including A. polyphaga) and Vermamoeba veriformis were the main FLA species present in both groundwater sites examined. While most of the isolated FLA co-existed with L. pneumophila, the FLA populations in the L. pneumophila co-culture experiments predominantly consisted of A. polyphaga, Acanthamoeba spp., Naegleria spp., V. vermiformis, Paravahlkampfia spp., and Echinamoeba spp. These observations suggest that FLA may have the potential to act as reservoirs for L. pneumophila in Canterbury, New Zealand groundwater systems and could be introduced into the local drinking water infrastructure, where they may promote the survival, multiplication, and dissemination of Legionella. This research addresses an important gap in our understanding of FLA-mediated pathogen dispersal in freshwater ecosystems.

We have recently developed new synthetic DNA tracers for tracking sources and pathways of contamination in surface water and groundwater. The use of DNA tracers in natural water systems results in substantial and rapid dilutions, thus accurate quantitation of initial DNA tracer concentrations applied is crucial to ensure their successful downstream detections. We compared the sensitivity and accuracy of three portable analytical techniques for quantitation of these DNA tracers: Nanodrop, Qubit, and OptiQ. All three methods were about equally effective when measuring high concentrations of DNA tracers (e.g., for c‐amine DNA tracer 1.54 × 105, 1.37 × 105, and 1.77 × 105 ng/mL for Nanodrop, Qubit, and OptiQ, respectively). However, the fluorescent methods of Qubit and OptiQ were significantly more sensitive at detecting lower concentrations of DNA tracers with limits of detection in the range 0.1–2 ng/mL, compared to 5 × 103 ng/mL for Nanodrop. The results of this work will facilitate the practical deployment of DNA tracers for tracking water contamination, and improving freshwater quality. Core Ideas DNA tracers are a novel technology to track water contamination. DNA tracers are rapidly diluted, so quantitation must be reliable. We evaluated three portable analytical technologies for quantitating DNA tracers. Fluorescent quantitation methods were found to be the most sensitive. These data will facilitate DNA tracer deployment for tracking water contamination.