Defeating Antibiotic- and Phage-Resistant Enterococcus faecalis Using a Phage Cocktail in Vitro and in a Clot Model. - Related Documents




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909001.0000Defeating Antibiotic- and Phage-Resistant Enterococcus faecalis Using a Phage Cocktail in Vitro and in a Clot Model. The deteriorating effectiveness of antibiotics is propelling researchers worldwide towards alternative techniques such as phage therapy: curing infectious diseases using viruses of bacteria called bacteriophages. In a previous paper, we isolated phage EFDG1, highly effective against both planktonic and biofilm cultures of one of the most challenging pathogenic species, the vancomycin-resistant Enterococcus (VRE). Thus, it is a promising phage to be used in phage therapy. Further experimentation revealed the emergence of a mutant resistant to EFDG1 phage: EFDG1(r). This kind of spontaneous resistance to antibiotics would be disastrous occurrence, however for phage-therapy it is only a minor hindrance. We quickly and successfully isolated a new phage, EFLK1, which proved effective against both the resistant mutant EFDG1(r) and its parental VRE, Enterococcus faecalis V583. Furthermore, combining both phages in a cocktail produced an additive effect against E. faecalis V583 strains regardless of their antibiotic or phage-resistance profile. An analysis of the differences in genome sequence, genes, mutations, and tRNA content of both phages is presented. This work is a proof-of-concept of one of the most significant advantages of phage therapy, namely the ability to easily overcome emerging resistant bacteria.201829541067
947010.9996Practical Method for Isolation of Phage Deletion Mutants. The growing concern about multi-drug resistant pathogenic bacteria has led to a renewed interest in the study of bacteriophages as antimicrobials and as therapeutic agents against infectious diseases (phage therapy). Phages to be used for this purpose have to be subjected to in-depth genomic characterization. It is essential to ascribe specific functions to phage genes, which will give information to unravel phage biology and to ensure the lack of undesirable genes, such as virulence and antibiotic resistance genes. Here, we describe a simple protocol for the selection of phage mutants carrying random deletions along the phage genome. Theoretically, any DNA region might be removed with the only requirement that the phage particle viability remains unaffected. This technique is based on the instability of phage particles in the presence of chelating compounds. A fraction of the phage population naturally lacking DNA segments will survive the treatment. Within the context of phages as antimicrobials, this protocol is useful to select lytic variants from temperate phages. In terms of phage efficiency, virulent phages are preferred over temperate ones to remove undesirable bacteria. This protocol has been used to obtain gene mutations that are involved in the lysogenic cycle of phages infecting Gram-positive bacteria (Staphylococcus and Lactobacillus).201831164553
950620.9996Nisin resistance in Gram-positive bacteria and approaches to circumvent resistance for successful therapeutic use. Antibiotic resistance among bacterial pathogens is one of the most worrying problems in health systems today. To solve this problem, bacteriocins from lactic acid bacteria, especially nisin, have been proposed as an alternative for controlling multidrug-resistant bacteria. Bacteriocins are antimicrobial peptides that have activity mainly against Gram-positive strains. Nisin is one of the most studied bacteriocins and is already approved for use in food preservation. Nisin is still not approved for human clinical use, but many in vitro studies have shown its therapeutic effectiveness, especially for the control of antibiotic-resistant strains. Results from in vitro studies show the emergence of nisin-resistant bacteria after exposure to nisin. Considering that nisin has shown promising results for clinical use, studies to elucidate nisin-resistant mechanisms and the development of approaches to circumvent nisin-resistance are important. Thus, the objectives of this review are to identify the Gram-positive bacterial strains that have shown resistance to nisin, describe their resistance mechanisms and propose ways to overcome the development of nisin-resistance for its successful clinical application.202133689548
909130.9995Characterization of an Enterococcus faecalis Bacteriophage vB_EfaM_LG1 and Its Synergistic Effect With Antibiotic. Enterococcus faecalis is a Gram-positive opportunistic pathogen that could cause pneumonia and bacteremia in stroke patients. The development of antibiotic resistance in hospital-associated E. faecalis is a formidable public health threat. Bacteriophage therapy is a renewed solution to treat antibiotic-resistant bacterial infections. However, bacteria can acquire phage resistance quite quickly, which is a significant barrier to phage therapy. Here, we characterized a lytic E. faecalis bacteriophage Vb_EfaM_LG1 with lytic activity. Its genome did not contain antibiotic resistance or virulence genes. Vb_EfaM_LG1 effectively inhibits E. faecalis growth for a short period, and phage resistance developed within hours. However, the combination of antibiotics and phage has a tremendous synergistic effect against E. faecalis, prevents the development of phage resistance, and disrupts the biofilm efficiently. Our results show that the phage-antibiotic combination has better killing efficiency against E. faecalis.202134336721
884940.9995Attenuating the Selection of Vancomycin Resistance Among Enterococci through the Development of Peptide-Based Vancomycin Antagonists. The emergence and spread of multidrug resistant (MDR) pathogens with acquired resistance to almost all available antimicrobial agents has severely threatened the international healthcare community over the last two decades. The last resort antibiotic vancomycin is critical for treatment of several of these pathogens; howeverc vancomycin resistance is spreading due to the undesired accumulation of IV vancomycin in the colon post-treatment. This accumulation exerts selective pressure upon members of the colonic microflora, including Enterococci, which possess vancomycin resistance genes. To ensure the continual effectiveness of vancomycin in the clinical setting by preventing the spread of antibiotic resistance, it is crucial to develop strategies that reduce selective pressure on the colonic microflora while allowing vancomycin to maintain its desired activity at the site of infection. Herein we report that modification of the native l-Lys-d-Ala-d-Ala vancomycin binding site can be used to produce peptides with the ability to competitively bind vancomycin, reducing its activity against susceptible Enterococci. Moreover, several modifications to the N-termini of the native tripeptide have produced compounds with enhanced vancomycin binding activity, including several analogs that were designed to covalently bind vancomycin, thereby acting as suicide inhibitors. Finally, in a mixed culture of susceptible and resistant bacteria, a single lead compound was found to protect high ratios of susceptible bacteria from vancomycin over the course of a week-long period, preventing the selection for vancomycin-resistant Enterococci. These findings demonstrate the ability of these peptides as potential therapeutic adjuvants for counteracting the undesired accumulation of colonic vancomycin, allowing for protection of the colonic microflora.202032946213
947350.9995The role of the animal host in the management of bacteriophage resistance during phage therapy. Multi-drug-resistant bacteria are associated with significantly higher morbidity and mortality. The possibilities for discovering new antibiotics are limited, but phage therapy - the use of bacteriophages (viruses infecting bacteria) to cure infections - is now being investigated as an alternative or complementary treatment to antibiotics. However, one of the major limitations of this approach lies in the antagonistic coevolution between bacteria and bacteriophages, which determines the ultimate success or failure of phage therapy. Here, we review the possible influence of the animal host on phage resistance and its consequences for the efficacy of phage therapy. We also discuss the value of in vitro assays for anticipating the dynamics of phage resistance observed in vivo.202336512896
940060.9995Conjugative Delivery of CRISPR-Cas9 for the Selective Depletion of Antibiotic-Resistant Enterococci. The innovation of new therapies to combat multidrug-resistant (MDR) bacteria is being outpaced by the continued rise of MDR bacterial infections. Of particular concern are hospital-acquired infections (HAIs) that are recalcitrant to antibiotic therapies. The Gram-positive intestinal pathobiont Enterococcus faecalis is associated with HAIs, and some strains are MDR. Therefore, novel strategies to control E. faecalis populations are needed. We previously characterized an E. faecalis type II CRISPR-Cas system and demonstrated its utility in the sequence-specific removal of antibiotic resistance determinants. Here, we present work describing the adaption of this CRISPR-Cas system into a constitutively expressed module encoded on a pheromone-responsive conjugative plasmid that efficiently transfers to E. faecalis for the selective removal of antibiotic resistance genes. Using in vitro competition assays, we show that these CRISPR-Cas-encoding delivery plasmids, or CRISPR-Cas antimicrobials, can reduce the occurrence of antibiotic resistance in enterococcal populations in a sequence-specific manner. Furthermore, we demonstrate that deployment of CRISPR-Cas antimicrobials in the murine intestine reduces the occurrence of antibiotic-resistant E. faecalis by several orders of magnitude. Finally, we show that E. faecalis donor strains harboring CRISPR-Cas antimicrobials are immune to uptake of antibiotic resistance determinants in vivo Our results demonstrate that conjugative delivery of CRISPR-Cas antimicrobials may be adaptable for future deployment from probiotic bacteria for exact targeting of defined MDR bacteria or for precision engineering of polymicrobial communities in the mammalian intestine.201931527030
422470.9995The Genus Enterococcus: Between Probiotic Potential and Safety Concerns-An Update. A considerable number of strains belonging to different species of Enterococcus are highly competitive due to their resistance to wide range of pH and temperature. Their competitiveness is also owed to their ability to produce bacteriocins recognized for their wide-range effectiveness on pathogenic and spoilage bacteria. Enterococcal bacteriocins have attracted great research interest as natural antimicrobial agents in the food industry, and as a potential drug candidate for replacing antibiotics in order to treat multiple drugs resistance pathogens. However, the prevalence of virulence factors and antibiotic-resistance genes and the ability to cause disease could compromise their application in food, human and animal health. From the current regulatory point of view, the genus Enterococcus is neither recommended for the QPS list nor have GRAS status. Although recent advances in molecular biology and the recommended methods for the safety evaluation of Enterococcus strains allowed the distinction between commensal and clinical clades, development of highly adapted methods and legislations are still required. In the present review, we evaluate some aspects of Enterococcus spp. related to their probiotic properties and safety concerns as well as the current and potential application in food systems and treatment of infections. The regulatory status of commensal Enterococcus candidates for food, feed, probiotic use, and recommended methods to assess and ensure their safety are also discussed.201830123208
961180.9995Parallel evolution of Pseudomonas aeruginosa phage resistance and virulence loss in response to phage treatment in vivo and in vitro. With rising antibiotic resistance, there has been increasing interest in treating pathogenic bacteria with bacteriophages (phage therapy). One limitation of phage therapy is the ease at which bacteria can evolve resistance. Negative effects of resistance may be mitigated when resistance results in reduced bacterial growth and virulence, or when phage coevolves to overcome resistance. Resistance evolution and its consequences are contingent on the bacteria-phage combination and their environmental context, making therapeutic outcomes hard to predict. One solution might be to conduct 'in vitro evolutionary simulations' using bacteria-phage combinations from the therapeutic context. Overall, our aim was to investigate parallels between in vitro experiments and in vivo dynamics in a human participant. Evolutionary dynamics were similar, with high levels of resistance evolving quickly with limited evidence of phage evolution. Resistant bacteria-evolved in vitro and in vivo-had lower virulence. In vivo, this was linked to lower growth rates of resistant isolates, whereas in vitro phage resistant isolates evolved greater biofilm production. Population sequencing suggests resistance resulted from selection on de novo mutations rather than sorting of existing variants. These results highlight the speed at which phage resistance can evolve in vivo, and how in vitro experiments may give useful insights for clinical evolutionary outcomes.202235188102
981590.9995Prospecting gene therapy of implant infections. Infection still represents one of the most serious and ravaging complications associated with prosthetic devices. Staphylococci and enterococci, the bacteria most frequently responsible for orthopedic postsurgical and implant-related infections, express clinically relevant antibiotic resistance. The emergence of antibiotic-resistant bacteria and the slow progress in identifying new classes of antimicrobial agents have encouraged research into novel therapeutic strategies. The adoption of antisense or "antigene" molecules able to silence or knock-out bacterial genes responsible for their virulence is one possible innovative approach. Peptide nucleic acids (PNAs) are potential drug candidates for gene therapy in infections, by silencing a basic gene of bacterial growth or by tackling the antibiotic resistance or virulence factors of a pathogen. An efficacious contrast to bacterial genes should be set up in the first stages of infection in order to prevent colonization of periprosthesis tissues. Genes encoding bacterial factors for adhesion and colonization (biofilm and/or adhesins) would be the best candidates for gene therapy. But after initial enthusiasm for direct antisense knock-out or silencing of essential or virulence bacterial genes, difficulties have emerged; consequently, new approaches are now being attempted. One of these, interference with the regulating system of virulence factors, such as agr, appears particularly promising.200919882546
9507100.9995Bacteriocins: Classification, synthesis, mechanism of action and resistance development in food spoilage causing bacteria. Huge demand of safe and natural preservatives has opened new area for intensive research on bacteriocins to unravel the novel range of antimicrobial compounds that could efficiently fight off the food-borne pathogens. Since food safety has become an increasingly important international concern, the application of bacteriocins from lactic acid bacteria that target food spoilage/pathogenic bacteria without major adverse effects has received great attention. Different modes of actions of these bacteriocins have been suggested and identified, like pore-forming, inhibition of cell-wall/nucleic acid/protein synthesis. However, development of resistance in the food spoilage and pathogenic bacteria against these bacteriocins is a rising concern. Emergence and spread of mutant strains resistant to bacteriocins is hampering food safety. It has spurred an interest to understand the bacteriocin resistance phenomenon displayed by the food pathogens, which will be helpful in mitigating the resistance problem. Therefore, present review is focused on the different resistance mechanisms adopted by food pathogens to overcome bacteriocin.201930610901
9508110.9995Nisin and class IIa bacteriocin resistance among Listeria and other foodborne pathogens and spoilage bacteria. Food safety has been an important issue globally due to increasing foodborne diseases and change in food habits. To inactivate foodborne pathogens, various novel technologies such as biopreservation systems have been studied. Bacteriocins are ribosomally synthesized peptides or proteins with antimicrobial activity produced by different groups of bacteria, but the bacteriocins produced by many lactic acid bacteria offer potential applications in food preservation. The use of bacteriocins in the food industry can help reduce the addition of chemical preservatives as well as the intensity of heat treatments, resulting in foods that are more naturally preserved. However, the development of highly tolerant and/or resistant strains may decrease the efficiency of bacteriocins as biopreservatives. Several mechanisms of bacteriocin resistance development have been proposed among various foodborne pathogens. The acquiring of resistance to bacteriocins can significantly affect physiological activity profile of bacteria, alter cell-envelope lipid composition, and also modify the antibiotic susceptibility/resistance profile of bacteria. This article presents a brief review on the scientific research about the various possible mechanisms involved in the development of resistance to nisin and Class IIa bacteriocins among the foodborne pathogens.201121417775
4061120.9994Beyond serial passages: new methods for predicting the emergence of resistance to novel antibiotics. Market launching of a new antibiotic requires knowing in advance its benefits and possible risks, and among them how rapidly resistance will emerge and spread among bacterial pathogens. This information is not only useful from a public health point of view, but also for pharmaceutical industry, in order to reduce potential waste of resources in the development of a compound that might be discontinued at the short term because of resistance development. Most assays currently used for predicting the emergence of resistance are based on culturing the target bacteria by serial passages in the presence of increasing concentrations of antibiotics. Whereas these assays may be valuable for identifying mutations that might cause resistance, they are not useful to establish how fast resistance might appear, neither to address the risk of spread of resistance genes by horizontal gene transfer. In this article, we review recent information pertinent for a more accurate prediction on the emergence and dispersal of antibiotic resistance.201121835695
9281130.9994Bacterial viruses enable their host to acquire antibiotic resistance genes from neighbouring cells. Prophages are quiescent viruses located in the chromosomes of bacteria. In the human pathogen, Staphylococcus aureus, prophages are omnipresent and are believed to be responsible for the spread of some antibiotic resistance genes. Here we demonstrate that release of phages from a subpopulation of S. aureus cells enables the intact, prophage-containing population to acquire beneficial genes from competing, phage-susceptible strains present in the same environment. Phage infection kills competitor cells and bits of their DNA are occasionally captured in viral transducing particles. Return of such particles to the prophage-containing population can drive the transfer of genes encoding potentially useful traits such as antibiotic resistance. This process, which can be viewed as 'auto-transduction', allows S. aureus to efficiently acquire antibiotic resistance both in vitro and in an in vivo virulence model (wax moth larvae) and enables it to proliferate under strong antibiotic selection pressure. Our results may help to explain the rapid exchange of antibiotic resistance genes observed in S. aureus.201627819286
4293140.9994Resistance to ocular antibiotics: an overview. The introduction of new antibiotic compounds into therapy initiates the development of resistance by the target bacteria. Resistance increases the risk of treatment failure with potentially serious consequences. Local application of antibacterial compounds to the eyes may lead to bacterial resistance in bacterial isolates from the eyes. The incidence of resistant strains of common pathogens is probably increasing. As compounds can be absorbed into the systemic circulation following ocular administration, the subsequent low concentrations in the blood could provide the selective pressure for the survival of resistant bacteria in the body. Despite this possibility, there are no reports of systemic resistance in bacteria following ocular administration of antibacterial compounds. All health-care professionals should be concerned about this possibility and continue to use these important compounds with respect.200717535364
4247150.9994Drug resistance in tuberculosis. Drug-resistant tuberculosis remains a worldwide problem. New laboratory methods have improved our ability to more rapidly identify resistant strains, but the most effective approach is to prevent the appearance of resistance by appropriate choice of antibiotics and directly-observed therapy. Mycobacterium tuberculosis is treated with familiar and unique drugs; consequently, mechanisms of resistance have some unique features. All drug resistance thus far identified develops by mutational events rather than acquisition of resistance genes from other bacteria. An agenda is presented for countering the appearance of further drug resistance in mycobacteria.19979421707
4249160.9994Detection of essential genes in Streptococcus pneumoniae using bioinformatics and allelic replacement mutagenesis. Although the emergence and spread of antimicrobial resistance in major bacterial pathogens for the past decades poses a growing challenge to public health, discovery of novel antimicrobial agents from natural products or modification of existing antibiotics cannot circumvent the problem of antimicrobial resistance. The recent development of bacterial genomics and the availability of genome sequences allow the identification of potentially novel antimicrobial agents. The cellular targets of new antimicrobial agents must be essential for the growth, replication, or survival of the bacterium. Conserved genes among different bacterial genomes often turn out to be essential (1, 2). Thus, the combination of comparative genomics and the gene knock-out procedure can provide effective ways to identify the essential genes of bacterial pathogens (3). Identification of essential genes in bacteria may be utilized for the development of new antimicrobial agents because common essential genes in diverse pathogens could constitute novel targets for broad-spectrum antimicrobial agents.200818392984
9431170.9994Biofilms and antimicrobial resistance. The pathogenesis of many orthopaedic infections is related to the presence of microorganisms in biofilms. I examine the emerging understanding of the mechanisms of biofilm-associated antimicrobial resistance. Biofilm-associated resistance to antimicrobial agents begins at the attachment phase and increases as the biofilm ages. A variety of reasons for the increased antimicrobial resistance of microorganisms in biofilms have been postulated and investigated. Although bacteria in biofilms are surrounded by an extracellular matrix that might physically restrict the diffusion of antimicrobial agents, this does not seem to be a predominant mechanism of biofilm-associated antimicrobial resistance. Nutrient and oxygen depletion within the biofilm cause some bacteria to enter a nongrowing (ie, stationary) state, in which they are less susceptible to growth-dependent antimicrobial killing. A subpopulation of bacteria might differentiate into a phenotypically resistant state. Finally, some organisms in biofilms have been shown to express biofilm-specific antimicrobial resistance genes that are not required for biofilm formation. Overall, the mechanism of biofilm-associated antimicrobial resistance seems to be multifactorial and may vary from organism to organism. Techniques that address biofilm susceptibility testing to antimicrobial agents may be necessary before antimicrobial regimens for orthopaedic prosthetic device-associated infections can be appropriately defined in research and clinical settings. Finally, a variety of approaches are being defined to overcome biofilm-associated antimicrobial resistance.200516056024
9469180.9994Reversing bacterial resistance to antibiotics by phage-mediated delivery of dominant sensitive genes. Pathogen resistance to antibiotics is a rapidly growing problem, leading to an urgent need for novel antimicrobial agents. Unfortunately, development of new antibiotics faces numerous obstacles, and a method that resensitizes pathogens to approved antibiotics therefore holds key advantages. We present a proof of principle for a system that restores antibiotic efficiency by reversing pathogen resistance. This system uses temperate phages to introduce, by lysogenization, the genes rpsL and gyrA conferring sensitivity in a dominant fashion to two antibiotics, streptomycin and nalidixic acid, respectively. Unique selective pressure is generated to enrich for bacteria that harbor the phages carrying the sensitizing constructs. This selection pressure is based on a toxic compound, tellurite, and therefore does not forfeit any antibiotic for the sensitization procedure. We further demonstrate a possible way of reducing undesirable recombination events by synthesizing dominant sensitive genes with major barriers to homologous recombination. Such synthesis does not significantly reduce the gene's sensitization ability. Unlike conventional bacteriophage therapy, the system does not rely on the phage's ability to kill pathogens in the infected host, but instead, on its ability to deliver genetic constructs into the bacteria and thus render them sensitive to antibiotics prior to host infection. We believe that transfer of the sensitizing cassette by the constructed phage will significantly enrich for antibiotic-treatable pathogens on hospital surfaces. Broad usage of the proposed system, in contrast to antibiotics and phage therapy, will potentially change the nature of nosocomial infections toward being more susceptible to antibiotics rather than more resistant.201222113912
4271190.9994Multi-step vs. single-step resistance evolution under different drugs, pharmacokinetics, and treatment regimens. The success of antimicrobial treatment is threatened by the evolution of drug resistance. Population genetic models are an important tool in mitigating that threat. However, most such models consider resistance emergence via a single mutational step. Here, we assembled experimental evidence that drug resistance evolution follows two patterns: (i) a single mutation, which provides a large resistance benefit, or (ii) multiple mutations, each conferring a small benefit, which combine to yield high-level resistance. Using stochastic modeling, we then investigated the consequences of these two patterns for treatment failure and population diversity under various treatments. We find that resistance evolution is substantially limited if more than two mutations are required and that the extent of this limitation depends on the combination of drug type and pharmacokinetic profile. Further, if multiple mutations are necessary, adaptive treatment, which only suppresses the bacterial population, delays treatment failure due to resistance for a longer time than aggressive treatment, which aims at eradication.202134001313