Isolation and genomic characterization of bacteriophages targeting extended-spectrum cephalosporin resistant E. coli
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Abstract:
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.Keywords:
Siphoviridae
Phage therapy
Myoviridae
Podoviridae
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Acinetobacter baumannii
Phage therapy
Enterococcus faecium
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Preliminary survey of local bacteriophages with lytic activity against multi‐drug resistant bacteria
Bacteriophages (phages) represent a potential alternative for combating multi‐drug resistant bacteria. Because of their narrow host range and the ever emergence of novel pathogen variants the continued search for phages is a prerequisite for optimal treatment of bacterial infections. Here we performed an ad hoc survey in the surroundings of a University hospital for the presence of phages with therapeutic potential. To this end, 16 aquatic samples of different origins and locations were tested simultaneously for the presence of phages with lytic activity against five current, but distinct strains each from the ESKAPE‐group (i.e., Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa , and Enterobacter cloacae ). Phages could be isolated for 70% of strains, covering all bacterial species except S. aureus . Apart from samples from two lakes, freshwater samples were largely devoid of phages. By contrast, one liter of hospital effluent collected at a single time point already contained phages active against two‐thirds of tested strains. In conclusion, phages with lytic activity against nosocomial pathogens are unevenly distributed across environments with the prime source being the immediate hospital vicinity.
Lytic cycle
Acinetobacter baumannii
Enterococcus faecium
Phage therapy
Enterobacter cloacae
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Worldwide, poultry industry suffers from infections caused by avian pathogenic Escherichia coli. Therapeutic failure due to resistant bacteria is of increasing concern and poses a threat to human and animal health. This causes a high demand to find alternatives to fight bacterial infections in animal farming. Bacteriophages are being especially considered for the control of multi-drug resistant bacteria due to their high specificity and lack of serious side effects. Therefore, the study aimed on characterizing phages and composing a phage cocktail suitable for the prevention of infections with E. coli. Six phages were isolated or selected from our collections and characterized individually and in combination with regard to host range, stability, reproduction, and efficacy in vitro. The cocktail consisting of six phages was able to inhibit formation of biofilms by some E. coli strains but not by all. Phage-resistant variants arose when bacterial cells were challenged with a single phage but not when challenged by a combination of four or six phages. Resistant variants arising showed changes in carbon metabolism and/or motility. Genomic comparison of wild type and phage-resistant mutant E28.G28R3 revealed a deletion of several genes putatively involved in phage adsorption and infection.
Phage therapy
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Abstract Background P. aeruginosa is the primary source of hospital-acquired infections. Unfortunately, antibiotic resistance is growing to precariously high levels, making the infections by this pathogen life-threatening and hard to cure. One possible alternative to antibiotics is to use phages. However, the isolation of phages suitable for phage therapy— be lytic, be efficient, and have a broad host range —against some target bacteria has proven difficult. To identify the best places to look for these phages against P. aeruginosa we screened hospital sewages, soils, and rivers in two cities. Results We isolated eighteen different phages, determined their host range, infection property, and plaque morphology. We found that the sewage and sewage-contaminated environments are the most reliable sources for the isolation of Pseudomonas phages. In addition, phages isolated from hospital sewage showed the highest efficiency in lysing the bacteria used for host range determination. In contrast, phages from the river had larger plaque size and lysed bacteria with higher levels of antibiotic resistance. Conclusions Our findings provided additional support for the importance of sewage as the source of phage isolation.
Phage therapy
Isolation
Lytic cycle
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The bacterial species of the Staphylococcus genus are important human and animal pathogens which cause severe infectious diseases. The most pathogenic species Staphylococcus aureus is the major causative agent of numerous hospital- and community-acquired infections. The increasing number of pathogenic strains resistant to antimicrobial drugs is a serious problem for public health that can be solved by phage therapy as a suitable alternative to antibiotics treatment. One of the disadvantages of phages is that compared to wide-spectrum antibiotics phages act on species or strains only. Using polyvalent bacteriophages or preparation of phage cocktails with broad host range is a suitable solution for this problem of fighting unwanted bacteria in medicine, food industry, biotechnology and agriculture. Two anti-staphylococcal phages MB401 and MB402 were chosen for a potential synergic use in a cocktail. Genomes of these phages were sequenced and alignment shows that they share more than 97% homology. Only few rearrangements in genome influenced host spectrum of these phages. Lytic spectrum of bacteriophages was tested on 53 clinical isolates of S. aureus from Czech hospitals including MRSA strains. 24 strains were sensitive due to synergic effect of both phages. In the future we are planning to isolate more phages which could extend host spectrum and add them into the phage cocktail. A number of analyses is required for commercial products especially in veterinary and human medicine. An important feature is declaration of nontoxicity of phage lysate. Phages MB401 and MB402 were propagated on well-defined bacterial strain which did not contain any known prophages, genes for toxins, virulence factors etc. and that increases the probability of a safe final product. Local dermal tolerance and acute dermal toxicity studies were performed with phage lysate on rats and rabbits and results confirm that phage lysate is nontoxic.
Lytic cycle
Prophage
Phage therapy
Human pathogen
Human medicine
Bacterial virus
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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.
Myoviridae
Phage therapy
Lytic cycle
Podoviridae
Siphoviridae
Lysogenic cycle
Phage typing
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The increase of multi-resistant bacteria highlights that the golden era of antibiotics is ending and that alternative treatmentsare urgently needed. Phages have been historically used to treat bacterial infections prior to the discovery of antibiotics and have gained renewed interest in the past decade. Despite the advantages of phage therapy over traditional antibiotic usage, a number of concerns persist over their clinical application centring on their efficacy and safety. This thesis presents four papers that focus on the isolation and characterization of phages that target reference strains and drug-resistant strains of E. coli as well as their infection dynamics and kinetics. In Paper I, six of thirty isolated phages were selected to be characterized for their growth parameters and host range using two commonly used methods. The study showed that the host range (an important selection criteria for phages) of the phages can change based on the assessment method and that the lysis efficiency of phages is host-dependent. The study suggests that standardised methods to assess the host range and lytic activity of phages are required to reduce result variability between research groups. Paper II investigated a rare phage with C3 morphotype from the Podoviridae family and characterised it via genomic, proteomic, morphologic and phylogenetic analysis. The study revealed previously unseen aspects including the formation of a honeycomb structure comprised of phage head during DNA packaging, the possible contractile nature of the tail and the 280 million year co-evolution between the major head protein and the scaffolding protein. Paper III highlights the need to take the immune system into consideration when designing phage therapeutics. In the study, four purified structurally distinct phages (selected from the three main phage families) were exposed to human cells (HT-29 and Caco-2 immortalised intestinal epithelial cell lines and donor-derived peripheral blood mononuclear cells) and the immunogenicity of the phages determined. Phage immunogenicity was shown to vary in a concentration and phage dependent manner with SU63 (a Myoviridae) being the most immunogenic phage and SU32 (a Siphoviridae) the least immunogenic. In the presence of human cells and a suitable host, phages were shown to maintain their killing efficacy as well as the ability to proliferate. Paper IV studies the infection dynamics of an experimental two-phage cocktail against a single bacterial host in vitro and in silico. However, in silico analysis and in vitro analysis produced conflicting results, in which mathematical modelling predicted the complete clearance of bacteria for all treatment scenarios whereas experimental results showed a 1-3log10 reduction in bacterial content. Practical experiments also showed increased anti-bacterial activity when the time between the additions of each phage was varied. This discrepancy suggests that the current mathematical model is unsuitable due to the inability to account for discrete variables such as interference.
Lytic cycle
Phage therapy
Podoviridae
Lysogenic cycle
Isolation
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Gram negative hospital acquired infection are a major problem since last two decades Acinetobacter baumanii has emerged as a highly trouble pathogen for many institutions globally. As a consequence of its immense ability to acquired antibiotic drug resistance determinant .It has justifiably been propelled to the fore front of scientific attention. Rapid spread of multidrug resistant isolate causing infection. Acinetobacter baumannii clinical strains, were selected from a new phage library for further characterization. The complete genomes of the two phages were analyzed. Both phages are characterized by broad host range and essential features of potential therapeutic phages, such as short latent period (27 and 21 min, respectively), high burst size (125 and 145, respectively), stability of activity in liquid culture and low frequency of occurrence of phage-resistant mutant bacterial cells. Genomic analysis showed that while bacteriophage with resemblance to some unclassified Pseudomonas aeruginosa phages, The newly isolated phages can serve as control A. baumannii infections.
Acinetobacter baumannii
Phage therapy
Opportunistic pathogen
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Antibiotic resistance is a common and serious public health worldwide. As an alternative to antibiotics, bacteriophage (phage) therapy offers one of the best solutions to antibiotic resistance. Bacteriophages survive where their bacterial hosts are found; thus, they exist in almost all environments and their applications are quite varied in the medical, environmental, and industrial fields. Moreover, a single phage or a mixture of phages can be used in phage therapy; mixed phages tend to be more effective in reducing the number and/or activity of pathogenic bacteria than that of a single phage.
Phage therapy
Pathogenic bacteria
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Lytic cycle
Siphoviridae
Phage therapy
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Citations (13)