Isolation and Characterization of Six Vibrio parahaemolyticus Lytic Bacteriophages From Seafood Samples
C.W. TanYaya RukayadiHanan HasanNoor-Azira Abdul-MutalibNuzul Noorahya JambariHirofumi HaraTze Young ThungEpeng LeeSon Radu
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Vibrio parahaemolyticus is a foodborne pathogen that is frequently isolated from a variety of seafood. To control this pathogenic Vibrio spp., the implementation of bacteriophages in aquaculture and food industries have shown a promising alternative to antibiotics. In this study, six bacteriophages isolated from the seafood samples demonstrated a narrow host range specificity that infecting only the V. parahaemolyticus strains. Morphological analysis revealed that bacteriophages Vp33, Vp22, Vp21, and Vp02 belong to the Podoviridae family, while bacteriophages Vp08 and Vp11 were categorized into the Siphoviridae family. All bacteriophages were composed of DNA genome and showed distinctive restriction fragment length polymorphism. The optimal MOI for bacteriophage propagation was determined to be 0.001 to 1. One-step growth curve revealed that the latent period ranged from 10 to 20 min, and the burst size of bacteriophage was approximately 17 to 51 PFU/cell. The influence of temperature and pH levels on the stability of bacteriophages showed that all bacteriophages were optimally stable over a wide range of temperatures and pH levels. In vitro lytic activity of all bacteriophages demonstrated to have a significant effect against V. parahaemolyticus . Besides, the application of a bacteriophage cocktail instead of a single bacteriophage suspension was observed to have a better efficiency to control the growth of V . parahaemolyticus . Results from this study provided a basic understanding of the physiological and biological properties of the isolated bacteriophages before it can be readily used as a biocontrol agent against the growth of V. parahaemolyticus .Keywords:
Lytic cycle
Siphoviridae
Podoviridae
Myoviridae
Phage therapy
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Phages drive bacterial diversity, profoundly influencing microbial communities, from microbiomes to the drivers of global biogeochemical cycling. Aiming to broaden our understanding of
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Abstract Background Nowadays, 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. Methods This 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. Results Studied 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. Conclusions The 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.
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Multi-drug resistant (MDR) clinical strains of Pseudomonas aeruginosa are the most prevalent bacteria in the lungs of patients with cystic fibrosis (CF) and burn wounds and among the most common in immunocompromised hospital patients in Australia. There are currently no promising antibiotics in the pipeline being developed against these strains. Phage therapy, which uses viruses known as bacteriophages to infect and kill pathogenic bacteria, could be a possible alternative treatment. To this end, we isolated and characterised four novel phages against Australian clinical strains of P. aeruginosa isolated from patients with cystic fibrosis, from infected blood and joint aspirate in Southeast Queensland, Australia. Activated sludge was enriched for phages using the clinical strains, and four bacteriophages were isolated. The phages were able to cause lysis in a further three identified clinical isolates. Morphology showed that they were all tailed phages (of the order Caudovirales), two belonging to the family Myoviridae and the others assigned to the Podoviridae and Siphoviridae. Their genomes were sequenced to reveal a doubled stranded DNA topology with genome sizes ranging from 42 kb to 65 kb. In isolating and characterising these novel phages, we directed our efforts toward the development and use of these phages as candidates for phage therapy as an alternative strategy for the management or elimination of these pathogenic strains. Here we describe novel phage candidates for potential therapeutic treatment of MDR Australian clinical isolates of P. aeruginosa.
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Abstract Carbapenemase-producing Enterobacteriaceae (CPE) are one of the most detrimental species of antibiotic-resistant bacteria worldwide. Phage therapy has emerged as an effective strategy for the treatment of infections caused by CPE pathogens. In west Japan, the increasing occurrence of Klebsiella pneumoniae harboring the pKPI-6 plasmid, which encodes bla IMP-6 , is a growing concern. To manage such major antimicrobial-resistant pathogens, we isolated 29 novel phages from sewage in Japan, targeting 31 strains of K. pneumoniae and one strain of Escherichia coli harboring the pKPI-6 plasmid. Electron microscopy analysis indicated that of the 29 isolated phages, 21 (72.4%), 5 (17.2%), and 3 (10.3%) belonged to Myoviridae, Siphoviridae , and Podoviridae , respectively. Host range analysis revealed that 20 Myoviridae members in isolated phages infected 25–26 strains of K. pneumoniae , indicating that most of the isolated phages have a broad host range. The K. pneumoniae Kp21 can only be infected by phage øKp_21, while Kp22 can be infected by more than 20 phages. We applied a phage cocktail, which consists of 10 phages, against Kp21 and Kp22 and found that the phage cocktail delayed the emergence of phage-resistant bacteria for Kp21 strain but not for the Kp22 strain. Furthermore, phage-resistant Kp21 (Kp21r) became prone to be infected from other bacteriophages as a “trade-off” of resistance to phage øKp_21. Our proposed phage set has an adequate number of phages to combat the K. pneumoniae strain isolated in Japan. Notably, our work demonstrates how a suitable phage cocktail diminishes the occurrence of phage-resistant bacteria. Importance Klebsiella pneumoniae harboring the plasmid carrying bla IMP-6 is becoming an increasingly hazardous species in Japan. We collected and characterized 29 novel bacteriophages that infect K. pneumoniae carrying the pKPI-6 plasmid, isolated in clinical settings of west Japan. Our phages showed broad host ranges. We applied a phage cocktail treatment constructed from 10 phages against two host strains, Kp21 and Kp22, which show different phage susceptibility patterns each other. Although the phage cocktail delayed phage-resistant Kp21 emergence, the emergence of phage-resistant Kp22 could not be delayed. Moreover, phage-resistant Kp21 became sensitive to other phages, which did not originally infect wild-type Kp21. Our study demonstrates how a suitable phage cocktail can diminish the occurrence of phage-resistant bacteria.
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Abstract Achromobacter xylosoxidans , an opportunistic pathogen, is responsible for various nosocomial and community-acquired infections. We isolated phiAxp-3, an N4-like bacteriophage that infects A. xylosoxidans , from hospital waste and studied its genomic and biological properties. Transmission electron microscopy revealed that, with a 67-nm diameter icosahedral head and a 20-nm non-contractile tail, phiAxp-3 has features characteristic of Podoviridae bacteriophages (order Caudovirales ). With a burst size of 9000 plaque-forming units and a latent period of 80 min, phiAxp-3 had a host range limited to only four A. xylosoxidans strains of the 35 strains that were tested. The 72,825 bp phiAxp-3 DNA genome, with 416-bp terminal redundant ends, contains 80 predicted open reading frames, none of which are related to virulence or drug resistance. Genome sequence comparisons place phiAxp-3 more closely with JWAlpha and JWDelta Achromobacter phages than with other N4 viruses. Using proteomics, we identified 25 viral proteins from purified phiAxp-3 particles. Notably, investigation of the phage phiAxp-3 receptor on the surface of the host cell revealed that lipopolysaccharide serves as the receptor for the adsorption of phage phiAxp-3. Our findings advance current knowledge about A. xylosoxidans phages in an age where alternative therapies to combat antibiotic-resistant bacteria are urgently needed.
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Phage therapy is the therapeutic use of bacteriophages to treat highly drug resistant bacterial infections. Klebsiella pneumonia (KP) has emerged as an increasingly important cause of community-acquired nosocomial infections and many of these strains are highly virulent with multidrug resistance (MDR) ability and exhibit a strong propensity to spread making infection cause by it difficult to treat. Treatment utilizing bacteriophage specific to KP (KP phage) can be used as an alternative counter measure. Characterization of bacteriophage is utmost important in assisting the application of bacteriophage in phage therapy. In the present study, samples from sewage water and cockles were screened for KP phage. Enrichment detection methods followed by isolation of phage by double agar layers method was done using 9 strains of MDR-KP as host system. KP phages were characterized on the basis of plaque morphology, host range and transmission electron microscope (TEM). A total of 58 lytic KP phages were isolated from sewage (42) and cockle (16). Investigation done on host range showed that 52 were KP phage and 6 have broad host range. Further identification by transmission electron microscopy (TEM) was done to 10 selected phages that showed potent lytic activity against MDR-KP. Image from TEM show selected phages were from subfamily caudovirales (7) with icosahedral head and pentagonal structure with short non-contractile tail suggesting it belongs to the family of Podoviridae. Two phages belonged to the family Myoviridae with isometric head and contractile tail and one from the family Siphoviridae with icosahedral and a non-contractile tail. Therefore, this study has partially characterized and revealed 10 potential lytic bacteriophages as therapeutic agent against MDR-KP which mostly resembles Podoviridae family.
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Overuse of antibiotics has resulted in the emergence of antibiotic resistant bacteria resulting in bacterial infections in livestock and humans, that can no longer be controlled by these drugs [2]. Third generation cephalosporins are an antibiotic class used in critical situations as the last line of defence, however bacteria have now developed resistance to these drugs [3].
Bacteriophages are viruses which can infect and destroy bacteria, and are being developed as a new therapeutic method for the control and management of bacterial infections in swine. This method offers a highly specific therapy with minimal side effects on the gut microflora [4]. Administration of phages in animal feed has resulted in a reduction of the severity of bacterial infections in addition to a reduction in the shedding of bacteria in faecal matter [2, 5]. This shedding is a major human health concern as it has the potential to transfer antibiotic resistant bacteria and plasmids carrying resistant genes to humans through the faecal to oral route.
This project isolated 21 bacteriophages, from three separate sources, that are capable of lysing extended-spectrum cephalosporin (ESC) resistant E. coli. Characterisation of these phages, through electron microscopy and genome sequencing, identified phages belonging to the three different families within the order Caudovirales; Siphoviridae, Myoviridae and Podoviridae. Analysis of the phage genomes resulted in the identification of two clusters within the phages belonging to the Siphoviridae family, named Cluster 1 and 2. Comparison of the specificity of phages sourced from pig farms with (South Australia) and without (Murdoch University) ESC resistant bacteria suggests that highly specific phages can be sourced from locations infected and uninfected by the target bacterial isolate. Three of the phages isolated from Murdoch University have a broad host range of the target ESC resistant E. coli isolates, highlighting these phages for further studies and potential development into therapeutic products.
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Abstract Escherichia coli is a common fecal coliform, facultative aerobic, gram-negative bacterium. Pathogenic strains of such microbes have evolved to cause diarrhea, urinary tract infections, and septicemias. The emergence of antibiotic resistance urged the identification of an alternative strategy. The use of lytic bacteriophages against the control of pathogenic E. coli in clinics and different environmental setups (waste and drink water management) has become an alternative therapy to antibiotic therapy. Thus, this study aimed to isolate and characterize lytic bacteriophage from various sources in Addis Ababa, tested them against antimicrobial-resistant diarrheagenic E. coli strains and evaluated their therapeutic potential under in vitro conditions. A total of 14 samples were processed against six different diarrheagenic E. coli strains. The conventional culture and plaque analysis agar overlay method was used to recover lytic bacteriophage isolates. The phage isolates were characterized to determine their lytic effect, growth characteristics, host range activity and stability under different temperature and pH conditions. Phage isolates were identified by scanning electron microscope (SEM), and molecular techniques (PCR). In total, 17 phages were recovered from 84 tested plates. Of the 17 phage isolates, 11 (65%) were Myoviridae -like phages, and 6 (35%) phage isolates were Podoviridae and Siphoviridae by morphology and PCR identification. Based on the host range test, growth characteristics and stability test 7 potent phages were selected. These phages demonstrated better growth characteristics, including short latent periods, highest burst sizes, and wider host ranges, as well as thermal stability and the ability to survive in a wide range of pH levels. The promising effect of these phages against AMR pathogens has raised the possibility of their use in the biological control of bacterial infections.
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Coliphage
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