Aidan Coffey

Evaluation of a Cocktail of Three Bacteriophages for Biocontrol of Escherichia coli O157:H7

ABSTRACT Escherichia coli O157:H7 is an endemic pathogen causing a variety of human diseases including mild diarrhea, hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura. This study concerns the exploitation of bacteriophages as biocontrol agents to eliminate the pathogen E. coli O157:H7. Two distinct lytic phages (e11/2 and e4/1c) isolated against a human strain of E. coli O157:H7, a previously isolated lytic phage (pp01), and a cocktail of all three phages were evaluated for their ability to lyse the bacterium in vivo and in vitro. Phage e11/2, pp01, and the cocktail of all three virulent phages resulted in a 5-log-unit reduction of pathogen numbers in 1 h at 37°C. However, bacteriophage-insensitive mutants (BIMs) emerged following the challenge. All tested BIMs had a growth rate which approximated that of the parental O157 strain, although many of these BIMs had a smaller, more coccoid cellular morphology. The frequency of BIM formation (10−6 CFU) was similar for e11/2, pp01, and the phage cocktail, while BIMs insensitive to e4/1c occurred at the higher frequency (10−4 CFU). In addition, BIMs commonly reverted to phage sensitivity within 50 generations. In an initial meat trial experiment, the phage cocktail completely eliminated E. coli O157:H7 from the beef meat surface in seven of nine cases. Given that the frequency of BIM formation is low (10−6 CFU) for two of the phages, allied to the propensity of these mutants to revert to phage sensitivity, we expect that BIM formation should not hinder the use of these phages as biocontrol agents, particularly since low levels of the pathogen are typically encountered in the environment.

Bacteriophage and their lysins for elimination of infectious bacteria

When phages were originally identified, the possibility of using them as antibacterial agents against pathogens was immediately recognized and put into practise based on the knowledge available at the time. However, with the advent of antibiotics a decline in the use of phage as therapeutics followed. Phages did, however, become more useful in the study of fundamental aspects of molecular biology and in the diagnostic laboratory for the identification of pathogenic bacteria. More recently, the original application of phage as therapeutics to treat human and animal infections has been rekindled, particularly in an era where antibiotic resistance has become so problematic/commonplace. Phage lysins have also been studied and utilized in their own right as potential therapeutics for the treatment of bacterial infections. Indeed the past decade has seen a considerable amount of research worldwide focused on the engineering of phages as antibacterial agents in a wide range of applications. Furthermore, the US Food and Drug Administration and/or the US Department of Agriculture have recently approved commercial phage preparations to prevent bacterial contamination of livestock, food crops, meat and other foods. Such developments have prompted this review into the status of phage research as it pertains to the control of infectious bacteria.

Potential of the Polyvalent Anti-Staphylococcus Bacteriophage K for Control of Antibiotic-Resistant Staphylococci from Hospitals

ABSTRACT The increasing prevalence of antibiotic-resistant staphylococci has prompted the need for antibacterial controls other than antibiotics. In this study, a lytic bacteriophage (phage K) was assessed in vitro for its ability to inhibit emerging drug-resistant Staphylococcus aureus strains from hospitals and other species of Staphylococcus isolated from bovine infections. In in vitro inhibitory assays, phage K lysed a range of clinically isolated methicillin-resistant S. aureus (MRSA) strains, S. aureus with heterogeneous vancomycin resistance and vancomycin resistance, and teicoplanin-resistant strains. In these assays, 14 of the MRSA strains were initially only weakly sensitive to this phage. However, propagation of phage K on these less-sensitive strains resulted in all 14 being sensitive to the modified phages. The results enforce the principle that, while certain target bacteria may be relatively insensitive to lytic phage, this can be overcome by obtaining modified phage variants from passage of the phage through the insensitive strains. Model in situ hand wash studies using a phage-enriched wash solution resulted in a 100-fold reduction in staphylococcal numbers on human skin by comparison with numbers remaining after washing in phage-free solution. Infusion of the phage into a nonimmunogenic bismuth-based cream resulted in strong anti-Staphylococcus activity from the cream on plates and in broth.

Genome of Staphylococcal Phage K: a New Lineage of Myoviridae Infecting Gram-Positive Bacteria with a Low G+C Content

Phage K is a polyvalent phage of the Myoviridae family which is active against a wide range of staphylococci. Phage genome sequencing revealed a linear DNA genome of 127,395 bp, which carries 118 putative open reading frames. The genome is organized in a modular form, encoding modules for lysis, structural proteins, DNA replication, and transcription. Interestingly, the structural module shows high homology to the structural module from Listeria phage A511, suggesting intergenus horizontal transfer. In addition, phage K exhibits the potential to encode proteins necessary for its own replisome, including DNA ligase, primase, helicase, polymerase, RNase H, and DNA binding proteins. Phage K has a complete absence of GATC sites, making it insensitive to restriction enzymes which cleave this sequence. Three introns (lys-I1, pol-I2, and pol-I3) encoding putative endonucleases were located in the genome. Two of these (pol-I2 and pol-I3) were found to interrupt the DNA polymerase gene, while the other (lys-I1) interrupts the lysin gene. Two of the introns encode putative proteins with homology to HNH endonucleases, whereas the other encodes a 270-amino-acid protein which contains two zinc fingers (CX(2)CX(22)CX(2)C and CX(2)CX(23)CX(2)C). The availability of the genome of this highly virulent phage, which is active against infective staphylococci, should provide new insights into the biology and evolution of large broad-spectrum polyvalent phages.

The Recombinant Phage Lysin LysK Has a Broad Spectrum of Lytic Activity against Clinically Relevant Staphylococci, Including Methicillin-Resistant Staphylococcus aureus

This study concerns the cloning, characterization, and expression of the lysin (LysK) from staphylococcal phage K in Lactococcus lactis. Lactococcal lysates containing recombinant LysK were found to inhibit a range of different species of staphylococci isolated from bovine and human infection sources, including methicillin-resistant Staphylococcus aureus. LysK thus has potential as an antimicrobial for applications in the prevention and/or treatment of infections caused by staphylococci.

Bacteriophage-resistance systems in dairy starter strains: molecular analysis to application

Starter inhibition by bacteriophage infection in dairy fermentations can limit the usage of specific bacterial strains used in the manufacture of Cheddar, Mozzarella and other cheeses and can result in substantial economic losses. A variety of practical measures to alleviate the problem of phage infection have been adopted over the years but has invariably resulted in a very limited number of strains which can withstand intensive usage in industry. The application of genetic techniques to improve the phage-resistance of starter cultures for dairy fermentations has been intensively studied for the last 20 years to a point where this approach now has significant potential to alleviate the problem. This paper highlights the recent findings and developments that have been described in the literature that will have an impact on improvement of the phage-resistance of starter cultures.

Movers and shakers: Influence of bacteriophages in shaping the mammalian gut microbiota

The human intestinal microbiota is one of the most densely populated ecosystems on Earth, containing up to 1013 bacteria/g and in some respects can be considered an organ itself given its role in human health. Bacteriophages (phages) are the most abundant replicating entities on the planet and thrive wherever their bacterial hosts exist. They undoubtedly influence the dominant microbial populations in many ecosystems including the human intestine. Within this setting, lysogeny appears to be the preferred life cycle, presumably due to nutrient limitations and lack of suitable hosts protected in biofilms, hence the predator/prey dynamic observed in many ecosystems is absent. On the other hand, free virulent phages in the gut are more common among sufferers of intestinal diseases and have been shown to increase with antibiotic usage. Many of these phages evolve from prophages of intestinal bacteria and emerge under conditions where their bacterial hosts encounter stress suggesting that prophages can significantly alter the microbial community composition. Based on these observations, we propose the “community shuffling” model which hypothesizes that prophage induction contributes to intestinal dysbiosis by altering the ratio of symbionts to pathobionts, enabling pathobiont niche reoccupation. The consequences of the increased phage load on the mammalian immune system are also addressed. While this is an area of intestinal biology which has received little attention, this review assembles evidence from the literature which supports the role of phages as one of the biological drivers behind the composition of the gut microbiota.

Recombinant bacteriophage lysins as antibacterials

With the increasing worldwide prevalence of antibiotic resistant bacteria, bacteriophage endolysins (lysins) represent a very promising novel alternative class of antibacterial in the fight against infectious disease. Lysins are phage-encoded peptidoglycan hydrolases which, when applied exogenously (as purified recombinant proteins) to Gram-positive bacteria, bring about rapid lysis and death of the bacterial cell. A number of studies have recently demonstrated the strong potential of these enzymes in human and veterinary medicine to control and treat pathogens on mucosal surfaces and in systemic infections. They also have potential in diagnostics and detection, bio-defence, elimination of food pathogens and control of phytopathogens. This review discusses the extensive research on recombinant bacteriophage lysins in the context of antibacterials, and looks forward to future development and potential.

Bacteriophages MR299-2 and NH-4 Can Eliminate Pseudomonas aeruginosa in the Murine Lung and on Cystic Fibrosis Lung Airway Cells

ABSTRACT Pseudomonas aeruginosa is a common cause of infection in the lungs of patients with cystic fibrosis (CF). In addition, biofilm formation and antibiotic resistance of Pseudomonas are major problems that can complicate antibiotic therapy. We evaluated the efficacy of using bacteriophages to kill the pathogen in both biofilms and in the murine lung. We isolated and characterized two phages from a local wastewater treatment plant, a myovirus (ϕNH-4) and a podovirus (ϕMR299-2). Both phages were active against clinical isolates of P. aeruginosa. Together, the two phages killed all 9 clinical isolate strains tested, including both mucoid and nonmucoid strains. An equal mixture of the two phages was effective in killing P. aeruginosa NH57388A (mucoid) and P. aeruginosa MR299 (nonmucoid) strains when growing as a biofilm on a cystic fibrosis bronchial epithelial CFBE41o- cell line. Phage titers increased almost 100-fold over a 24-h period, confirming replication of the phage. Furthermore, the phage mix was also effective in killing the pathogen in murine lungs containing 1 × 107 to 2 × 107 P. aeruginosa. Pseudomonas was effectively cleared (reduced by a magnitude of at least 3 to 4 log units) from murine lungs in 6 h. Our study demonstrates the efficacy of these two phages in killing clinical Pseudomonas isolates in the murine lung or as a biofilm on a pulmonary cell line and supports the growing interest in using phage therapy for the control and treatment of multidrug-resistant Pseudomonas lung infections in CF patients. IMPORTANCE Given the rise in antibiotic resistance, nonantibiotic therapies are required for the treatment of infection. This is particularly true for the treatment of Pseudomonas infection in patients with cystic fibrosis. We have identified two bacterial viruses (bacteriophages) that can kill Pseudomonas growing on human lung cells and in an animal model of lung infection. The use of bacteriophages is particularly appropriate because the killing agent can replicate on the target cell, generating fresh copies of the bacteriophage. Thus, in the presence of a target, the killing agent multiplies. By using two bacteriophages we can reduce the risk of resistant colonies developing at the site of infection. Bacteriophage therapy is an exciting field, and this study represents an important demonstration of efficacy in validated infection models. Given the rise in antibiotic resistance, nonantibiotic therapies are required for the treatment of infection. This is particularly true for the treatment of Pseudomonas infection in patients with cystic fibrosis. We have identified two bacterial viruses (bacteriophages) that can kill Pseudomonas growing on human lung cells and in an animal model of lung infection. The use of bacteriophages is particularly appropriate because the killing agent can replicate on the target cell, generating fresh copies of the bacteriophage. Thus, in the presence of a target, the killing agent multiplies. By using two bacteriophages we can reduce the risk of resistant colonies developing at the site of infection. Bacteriophage therapy is an exciting field, and this study represents an important demonstration of efficacy in validated infection models.

Phage Therapy in the Food Industry

Despite advances in modern technologies, the food industry is continuously challenged with the threat of microbial contamination. The overuse of antibiotics has further escalated this problem, resulting in the increasing emergence of antibiotic-resistant foodborne pathogens. Efforts to develop new methods for controlling microbial contamination in food and the food processing environment are extremely important. Accordingly, bacteriophages (phages) and their derivatives have emerged as novel, viable, and safe options for the prevention, treatment, and/or eradication of these contaminants in a range of foods and food processing environments. Whole phages, modified phages, and their derivatives are discussed in terms of current uses and future potential as antimicrobials in the traditional farm-to-fork context, encompassing areas such as primary production, postharvest processing, biosanitation, and biodetection. The review also presents some safety concerns to ensure safe and effective exploitation of bacteriophages in the future.