Long-term effects of single and combined introductions of antibiotics and bacteriophages on populations of Pseudomonas aeruginosa.
ABSTRACT: With escalating resistance to antibiotics, there is an urgent need to develop alternative therapies against bacterial pathogens and pests. One of the most promising is the employment of bacteriophages (phages), which may be highly specific and evolve to counter antiphage resistance. Despite an increased understanding of how phages interact with bacteria, we know very little about how their interactions may be modified in antibiotic environments and, reciprocally, how phage may affect the evolution of antibiotic resistance. We experimentally evaluated the impacts of single and combined applications of antibiotics (different doses and different types) and phages on in vitro evolving populations of the opportunistic pathogen Pseudomonas aeruginosa PAO1. We also assessed the effects of past treatments on bacterial virulence in vivo, employing larvae of Galleria mellonella to survey the treatment consequences for the pathogen. We find a strong synergistic effect of combining antibiotics and phages on bacterial population density and in limiting their recovery rate. Our long-term study establishes that antibiotic dose is important, but that effects are relatively insensitive to antibiotic type. From an applied perspective, our results indicate that phages can contribute to managing antibiotic resistance levels, with limited consequences for the evolution of bacterial virulence.
Project description:Temperate phages are bacterial viruses that as part of their life cycle reside in the bacterial genome as prophages. They are found in many species including most clinical strains of the human pathogens, Staphylococcus aureus and Salmonella enterica serovar Typhimurium. Previously, temperate phages were considered as only bacterial predators, but mounting evidence point to both antagonistic and mutualistic interactions with for example some temperate phages contributing to virulence by encoding virulence factors. Here we show that generalized transduction, one type of bacterial DNA transfer by phages, can create conditions where not only the recipient host but also the transducing phage benefit. With antibiotic resistance as a model trait we used individual-based models and experimental approaches to show that antibiotic susceptible cells become resistant to both antibiotics and phage by i) integrating the generalized transducing temperate phages and ii) acquiring transducing phage particles carrying antibiotic resistance genes obtained from resistant cells in the environment. This is not observed for non-generalized transducing temperate phages, which are unable to package bacterial DNA, nor for generalized transducing virulent phages that do not form lysogens. Once established, the lysogenic host and the prophage benefit from the existence of transducing particles that can shuffle bacterial genes between lysogens and for example disseminate resistance to antibiotics, a trait not encoded by the phage. This facilitates bacterial survival and leads to phage population growth. We propose that generalized transduction can function as a mutualistic trait where temperate phages cooperate with their hosts to survive in rapidly-changing environments. This implies that generalized transduction is not just an error in DNA packaging but is selected for by phages to ensure their survival.
Project description:Enteropathogenic Escherichia coli (EPEC) is a major pathogen for diarrheal diseases among children. Antibiotics, when used appropriately, are effective; however, their overuse and misuse have led to the rise of antibiotic resistance worldwide. Thus, there are renewed efforts into the development of phage therapy as an alternative antibacterial therapy. Because EPEC in vivo models have shortcomings, a surrogate is used to study the mouse pathogen Citrobacter rodentium in animal models. In this study, two new phages CrRp3 and CrRp10, which infect C. rodentium, were isolated and characterized. CrRp3 was found to be a new species within the genus Vectrevirus, and CrRp10 is a new strain within the species Escherichia virus Ime09, in the genus Tequatrovirus. Both phages appear to have independently evolved from E. coli phages, rather than other Citrobacter spp. phages. Neither phage strain carries known genes associated with bacterial virulence, antibiotic resistance, or lysogeny. CrRp3 is more potent, having a 24-fold faster adsorption rate and shorter lytic cycle when compared to the same properties of CrRp10. However, a lysis curve analysis revealed that CrRp10 prevented growth of C. rodentium for 18 h, whereas resistance developed against CrRp3 within 9 h. We also show that hypoxic (5% oxygen) conditions decreased CrRp3 ability to control bacterial densities in culture. In contrast, low oxygen conditions did not affect CrRp10 ability to replicate on C. rodentium. Together, CrRp10 is likely to be the better candidate for future phage therapy investigations.
Project description:Carbapenem-resistant Acinetobacter baumannii (CRAB) which is noted as a major pathogen associated with healthcare-associated infections has steadily developed beyond antibiotic control. Lytic bacteriophages with the characteristics of infecting and lysing specific bacteria have been used as a potential alternative to traditional antibiotics to solve multidrug-resistant bacterial infections. Here, we isolated A. baumannii-specific lytic phages and evaluated their potential therapeutic effect against lung infection caused by CRAB clinical strains. The combined lysis spectrum of four lytic phages' ranges was 87.5% (42 of 48) against CRAB clinical isolates. Genome sequence and analysis indicated that phage SH-Ab15519 is a novel phage which does not contain the virulence or antibiotic resistance genes. In vivo study indicated that phage SH-Ab15519 administered intranasally can effectively rescue mice from lethal A. baumannii lung infection without deleterious side effects. Our work explores the potential use of phages as an alternative therapeutic agent against the lung infection caused by CRAB strains.
Project description:The evolution of antibiotic resistance in bacteria is a global concern and the use of bacteriophages alone or in combined therapies is attracting increasing attention as an alternative. Evolutionary theory predicts that the probability of bacterial resistance to both phages and antibiotics will be lower than to either separately, due for example to fitness costs or to trade-offs between phage resistance mechanisms and bacterial growth. In this study, we assess the population impacts of either individual or combined treatments of a bacteriophage and streptomycin on the nosocomial pathogen Pseudomonas aeruginosa. We show that combining phage and antibiotics substantially increases bacterial control compared to either separately, and that there is a specific time delay in antibiotic introduction independent of antibiotic dose, that minimizes both bacterial density and resistance to either antibiotics or phage. These results have implications for optimal combined therapeutic approaches.
Project description:The evolution of multi-antibiotic resistance in bacterial pathogens, often resulting from de novo mutations, is creating a public health crisis. Phages show promise for combating antibiotic-resistant bacteria, the efficacy of which, however, may also be limited by resistance evolution. Here, we suggest that phages may be used as supplements to antibiotics in treating initially sensitive bacteria to prevent resistance evolution, as phages are unaffected by most antibiotics and there should be little cross-resistance to antibiotics and phages. In vitro experiments using the bacterium Pseudomonas fluorescens, a lytic phage, and the antibiotic kanamycin supported this prediction: an antibiotic-phage combination dramatically decreased the chance of bacterial population survival that indicates resistance evolution, compared with antibiotic treatment alone, whereas the phage alone did not affect bacterial survival. This effect of the combined treatment in preventing resistance evolution was robust to immigration of bacteria from an untreated environment, but not to immigration from environment where the bacteria had coevolved with the phage. By contrast, an isogenic hypermutable strain constructed from the wild-type P. fluorescens evolved resistance to all treatments regardless of immigration, but typically suffered very large fitness costs. These results suggest that an antibiotic-phage combination may show promise as an antimicrobial strategy.
Project description:The increasing threat of pathogen resistance to antibiotics requires the development of novel antimicrobial strategies. Here we present a proof of concept for a genetic strategy that aims to sensitize bacteria to antibiotics and selectively kill antibiotic-resistant bacteria. We use temperate phages to deliver a functional clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system into the genome of antibiotic-resistant bacteria. The delivered CRISPR-Cas system destroys both antibiotic resistance-conferring plasmids and genetically modified lytic phages. This linkage between antibiotic sensitization and protection from lytic phages is a key feature of the strategy. It allows programming of lytic phages to kill only antibiotic-resistant bacteria while protecting antibiotic-sensitized bacteria. Phages designed according to this strategy may be used on hospital surfaces and hand sanitizers to facilitate replacement of antibiotic-resistant pathogens with sensitive ones.
Project description:Bacteriophages have shown promise as therapeutic alternatives to antibiotics for the control of infectious bacteria, including the human pathogen Salmonella. However, the development of effective phage-based applications requires the elucidation of key interactions between phages and target hosts, particularly since host resistance to phage is inevitable. Little is known about the alteration of host phenotypes following the development of resistance to phage. The aim of this study is to evaluate the antibiotic susceptibility and virulence of a Salmonella isolate following the development of resistance to bacteriophage SI1. We observed enhanced susceptibility to tetracycline and decreased invasion capacity in a differentiated Caco-2 intestinal cell line. Whole genome sequence analysis revealed an array of mutations, most notably, truncations in vgrG1_2, a core gene involved in Type VI secretion and mutations in the lipopolysaccharide, thereby indicating the plausible attachment site of phage SI1. These findings shed light on understanding the underlying mechanism for phage immunity within the host. Importantly, we reveal an associated genetic cost to the bacterial host with developing resistance to phages. Taken together, these results will aid in advancing strategies to delay or eliminate the development of host resistance when designing informed phage-based antimicrobials.
Project description:The rising antibiotic resistance of bacteria imposes a severe threat on human health. Inhibition of bacterial virulence is an alternative approach to develop new antimicrobials. Molecules targeting antibiotic resistant enzymes have been used in combination with cognate antibiotics. It might be ideal that a molecule can simultaneously suppress virulence factors and antibiotic resistance. Here we combined genetic and computer-aided inhibitor screening to search for such molecules against the bacterial pathogen Pseudomonas aeruginosa. To identify target proteins that control both virulence and antibiotic resistance, we screened for mutants with defective cytotoxicity and biofilm formation from 93 transposon insertion mutants previously reported with increased antibiotic susceptibility. A pyrD mutant displayed defects in cytotoxicity, biofilm formation, quorum sensing and virulence in an acute mouse pneumonia model. Next, we employed a computer-aided screening to identify potential inhibitors of the PyrD protein, a dihydroorotate dehydrogenase (DHODase) involved in pyrimidine biosynthesis. One of the predicted inhibitors was able to suppress the enzymatic activity of PyrD as well as bacterial cytotoxicity, biofilm formation and antibiotic resistance. A single administration of the compound reduced the bacterial colonization in the acute mouse pneumonia model. Therefore, we have developed a strategy to identify novel treatment targets and antimicrobial molecules.
Project description:Staphylococcus aureus is a very successful opportunistic pathogen capable of causing a variety of diseases ranging from mild skin infections to life-threatening sepsis, meningitis and pneumonia. Its ability to display numerous virulence mechanisms matches its skill to display resistance to several antibiotics, including ?-lactams, underscoring the fact that new anti-S. aureus drugs are urgently required. In this scenario, the utilization of lytic bacteriophages that kill bacteria in a genus -or even species- specific way, has become an attractive field of study. In this report, we describe the isolation, characterization and sequencing of phages capable of killing S. aureus including methicillin resistant (MRSA) and multi-drug resistant S. aureus local strains from environmental, animal and human origin. Genome sequencing and bio-informatics analysis showed the absence of genes encoding virulence factors, toxins or antibiotic resistance determinants. Of note, there was a high similarity between our set of phages to others described in the literature such as phage K. Considering that reported phages were obtained in different continents, it seems plausible that there is a commonality of genetic features that are needed for optimum, broad host range anti-staphylococcal activity of these related phages. Importantly, the high activity and broad host range of one of our phages underscores its promising value to control the presence of S. aureus in fomites, industry and hospital environments and eventually on animal and human skin. The development of a cocktail of the reported lytic phages active against S. aureus-currently under way- is thus, a sensible strategy against this pathogen.
Project description:The emergence of antibiotic resistance in bacteria is a global concern. The use of bacteriophages (or phages) alone or combined with antibiotics is consolidating itself as an alternative approach to inactivate antibiotic-resistant bacteria. However, phage-resistant mutants have been considered as a major threat when phage treatment is employed. Escherichia coli is one of the main responsible pathogens for moderate and serious infections in hospital and community environments, being involved in the rapid evolution of fluoroquinolones and third-generation cephalosporin resistance. The aim of this study was to evaluate the effect of combined treatments of phages and antibiotics in the inactivation of E. coli. For this, ciprofloxacin at lethal and sublethal concentrations was added at different times (0, 6, 12 and 18 h) and was tested in combination with the phage ELY-1 to inactivate E. coli. The efficacy of the combined treatment varied with the antibiotic concentration and with the time of antibiotic addition. The combined treatment prevented bacterial regrowth when the antibiotic was used at minimum inhibitory concentration (MIC) and added after 6 h of phage addition, causing less bacterial resistance than phage and antibiotic applied alone (4.0 × 10-7 for the combined treatment, 3.9 × 10-6 and 3.4 × 10-5 for the antibiotics and the phages alone, respectively). Combined treatment with phage and antibiotic can be effective in reducing the bacterial density and it can also prevent the emergence of resistant variants. However, the antibiotic concentration and the time of antibiotic application are essential factors that need to be considered in the combined treatment.