RESUMEN
Multidrug-resistant pathogens are now thought to be the primary global causes of disease and death. Therefore, it is imperative to develop new effective bioactive compounds from microbial sources, such as Streptomyces species. Nevertheless, the pharmaceutical industry suffered financial losses and low-quality end products as a result of Streptomyces bacteriophage contamination. To reduce the likelihood of phage-induced issues in the medical industry, it is crucial to develop a method for finding phage-resistant strains. Hence, we aimed to isolate and characterize Streptomyces spp. and Streptomyces phages from various rhizospheric soil samples in Egypt and to investigate their antibacterial activities. Moreover, we targeted development of a Streptomyces phage-resistant strain to extract its active metabolites and further testing its antibacterial activity. Herein, the antibacterial activities of the isolated 58 Streptomyces isolates showed that 10 (17.2 %) Streptomyces isolates had antibacterial activities against the tested bacteria including Listeria monocytogenes, E. coli O157, Acinetobacter baumannii, methicillin resistant-vancomycin-intermediate Staphylococcus aureus (MRSA-VISA) and Micrococcus luteus. Three lytic bacteriophages (ÏPRSC1, ÏPRSC2, and ÏPRSC4) belonging to the families Siphoviridae and Podoviridae were obtained from the rhizospheric soil samples using the most potent S. abietis isolate as the host strain. The three isolated Streptomyces phages were thermostable, ultraviolet stable, infectious, and had a wide range of hosts against the 10 tested Streptomyces isolates with antibacterial activities. The DNA of the ÏPRSC1 and ÏPRSC4 phages were resistant to digestion by EcoRI and HindIII, but the DNA of ÏPRSC2 was resistant to digestion by EcoRI and sensitive to digestion by HindIII. Of note, we developed a S. abietis strain resistant to the three isolated phages and its antibacterial activities were twice that of the wild strain. Finally, telomycin was recognized as an antibacterial metabolite extracted from phage-resistant S. abietis strain, which was potent against the tested Gram-positive bacteria including L. monocytogenes, MRSA-VISA, and M. luteus. Thus, our findings open new horizons for researching substitute antimicrobial medications for both existing and reemerging illnesses.
Asunto(s)
Antibacterianos , Microbiología del Suelo , Streptomyces , Streptomyces/virología , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Egipto , Podoviridae/aislamiento & purificación , Siphoviridae/aislamiento & purificación , Siphoviridae/genética , Bacteriófagos/aislamiento & purificación , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/virología , Listeria monocytogenes/efectos de los fármacos , Listeria monocytogenes/virología , Micrococcus luteus/efectos de los fármacos , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/virología , Aminoglicósidos/farmacología , Pruebas de Sensibilidad Microbiana , RizosferaRESUMEN
Double-layer agar (DLA) overlay plaque assay is the gold standard for phage enumeration. However, it is cumbersome and time-consuming. Given the great interest in phage therapy, we explored alternative assays for phage quantitation. A total of 16 different phages belonging to Myoviridae, Siphoviridae, and Podoviridae families were quantitated with five K. pneumoniae, eight P. aeruginosa, and three A. baumannii host isolates. Phages were quantitated with the standard DLA assay (10 mL of LB soft agar 0.7% on LB hard agar 1.5%) and the new single-layer agar (SLA) assay (10 mL of LB soft agar 0.7%) with phages spread (spread) into or spotted (spot) onto soft agar. Phage concentrations with each assay were correlated with the standard assay, and the relative and absolute differences between each assay and the standard double-layer agar spread were calculated. Phage concentrations 1 × 104-8.3 x1012 PFU/mL with the standard DLA assay were quantitated with SLA-spread, SLA-spot, and DLA-spot assays, and the median (range) relative and absolute differences were <10% and <0.98 log10PFU/mL, respectively, for all phage/bacterial species (ANOVA P = 0.1-0.43), and they were highly correlated (r > 0.77, P < 0.01). Moreover, plaques could be quantified at 37°C after 4-h incubation for K. pneumoniae phages and 6-h incubation for P. aeruginosa and A. baumannii phages, and estimated concentrations remained the same over 24 hours. Compared to DLA assay, the SLA-spot assay required less media, it was 10 times faster, and generated same-day results. The SLA-spot assay was cheaper, faster, easier to perform, and generated similar phage concentrations as the standard DLA-spread assay.
Asunto(s)
Bacteriófagos , Bacteriófagos/aislamiento & purificación , Acinetobacter baumannii/virología , Pseudomonas aeruginosa/virología , Humanos , Ensayos Analíticos de Alto Rendimiento/métodos , Farmacorresistencia Bacteriana Múltiple , Carga Viral/métodos , Klebsiella pneumoniae/virología , Podoviridae/aislamiento & purificación , Myoviridae/aislamiento & purificación , Myoviridae/clasificación , Siphoviridae/aislamiento & purificación , Siphoviridae/clasificaciónRESUMEN
The phage lysin field has done nothing but grow in the last decades. As a result, many different research groups around the world are contributing to the field, often with certain methodological differences that pose a challenge to the interpretation and comparison of results. In this work, we present the case study of three Acinetobacter baumannii-targeting phage lysins (wild-type endolysin LysMK34 plus engineered lysins eLysMK34 and 1D10) plus one lysin with broad activity against Gram-positive bacteria (PlySs2) to provide exemplary evidence on the risks of generalization when using one of the most common lysin evaluation assays: the killing assay with resting cells. To that end, we performed killing assays with the aforementioned lysins using hypo-, iso- and hypertonic buffers plus human serum either as the reaction or the dilution medium in a systematic manner. Our findings stress the perils of creating hypotonic conditions or a hypotonic shock during a killing assay, suggesting that hypotonic buffers should be avoided as a test environment or as diluents before plating to avoid overestimation of the killing effect in the assayed conditions. As a conclusion, we suggest that the nature of both the incubation and the dilution buffers should be always clearly identified when reporting killing activity data, and that for experimental consistency the same incubation buffer should be used as a diluent for posterior serial dilution and plating unless explicitly required by the experimental design. In addition, the most appropriate buffer mimicking the final application must be chosen to obtain relevant results.
Asunto(s)
Acinetobacter baumannii , Bacteriófagos , Bacteriófagos/química , Bacteriófagos/fisiología , Bacteriófagos/genética , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/virología , Concentración Osmolar , Viabilidad Microbiana/efectos de los fármacos , Tampones (Química) , Humanos , Proteínas Virales/genética , Proteínas Virales/metabolismo , Proteínas Virales/química , Endopeptidasas/metabolismo , Endopeptidasas/químicaRESUMEN
Acinetobacter baumannii, which is resistant to multiple drugs, is an opportunistic pathogen responsible for severe nosocomial infections. With no antibiotics available, phages have obtained clinical attention. However, since immunocompromised patients are often susceptible to infection, the appropriate timing of administration is particularly important. During this research, we obtained a lytic phage vB_AbaM_P1 that specifically targets A. baumannii. We then assessed its potential as a prophylactic treatment for lung infections caused by clinical strains. The virus experiences a period of inactivity lasting 30 min and produces approximately 788 particles during an outbreak. Transmission electron microscopy shows that vB_AbaM_P1 was similar to the Saclayvirus. Based on the analysis of high-throughput sequencing and bioinformatics, vB_AbaM_P1 consists of 107537 bases with a G + C content of 37.68%. It contains a total of 177 open reading frames and 14 tRNAs. No antibiotic genes were detected. In vivo experiments, using a cyclophosphamide-induced neutrophil deficiency model, tested the protective effect of phage on neutrophil-deficient rats by prophylactic application of phage. The use of phages resulted in a decrease in rat mortality caused by A. baumannii and a reduction in the bacterial burden in the lungs. Histologic examination of lung tissue revealed a decrease in the presence of immune cells. The presence of phage vB_AbaM_P1 had a notable impact on preventing A. baumannii infection, as evidenced by the decrease in oxidative stress in lung tissue and cytokine levels in serum. Our research offers more robust evidence for the early utilization of bacteriophages to mitigate A. baumannii infection. KEY POINTS: â¢A novel Saclayvirus phage infecting A. baumannii was isolated from sewage. â¢The whole genome was determined, analyzed, and compared to other phages. â¢Assaying the effect of phage in preventing infection in neutrophil-deficient models.
Asunto(s)
Infecciones por Acinetobacter , Acinetobacter baumannii , Bacteriófagos , Genoma Viral , Acinetobacter baumannii/virología , Acinetobacter baumannii/genética , Animales , Infecciones por Acinetobacter/prevención & control , Infecciones por Acinetobacter/microbiología , Bacteriófagos/genética , Bacteriófagos/aislamiento & purificación , Bacteriófagos/fisiología , Ratas , Terapia de Fagos/métodos , Composición de Base , Modelos Animales de Enfermedad , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Pulmón/virología , Pulmón/microbiología , Neumonía/prevención & control , Neumonía/microbiología , Neumonía/virología , MasculinoRESUMEN
Filamentous bacteriophages belonging to the order Tubulavirales, family Inoviridae, significantly affect the properties of Gram-negative bacteria, but filamentous phages of many important pathogens have not been described so far. The aim of this study was to examine A. baumannii filamentous phages for the first time and to determine their effect on bacterial virulence. The filamentous phages were detected in 15.3% of A. baumannii strains as individual prophages in the genome or as tandem repeats, and a slightly higher percentage was detected in the culture collection (23.8%). The phylogenetic analyses revealed 12 new genera within the Inoviridae family. Bacteriophages that were selected and isolated showed structural and genomic characteristics of the family and were unable to form plaques. Upon host infection, these phages did not significantly affect bacterial twitching motility and capsule production but significantly affected growth kinetics, reduced biofilm formation, and increased antibiotic sensitivity. One of the possible mechanisms of reduced resistance to antibiotics is the observed decreased expression of efflux pumps after infection with filamentous phages.
Asunto(s)
Acinetobacter baumannii , Biopelículas , Genoma Viral , Filogenia , Acinetobacter baumannii/virología , Acinetobacter baumannii/genética , Biopelículas/crecimiento & desarrollo , Inovirus/genética , Inovirus/fisiología , Inovirus/aislamiento & purificación , Especificidad del Huésped , Antibacterianos/farmacología , Virulencia , Bacteriófagos/genética , Bacteriófagos/aislamiento & purificación , Bacteriófagos/fisiología , Bacteriófagos/clasificación , Profagos/genética , Profagos/fisiologíaRESUMEN
Acinetobacter baumannii, an opportunistic pathogen, poses a significant threat in intensive care units, leading to severe nosocomial infections. The rise of multi-drug-resistant strains, particularly carbapenem-resistant A. baumannii, has created formidable challenges for effective treatment. Given the prolonged development cycle and high costs associated with antibiotics, phages have garnered clinical attention as an alternative for combating infections caused by drug-resistant bacteria. However, the utilization of phage therapy encounters notable challenges, including the narrow host spectrum, where each phage targets a limited subset of bacteria, increasing the risk of phage resistance development. Additionally, uncertainties in immune system dynamics during treatment hinder tailoring symptomatic interventions based on patient-specific states. In this study, we isolated two A. baumannii phages from wastewater and conducted a comprehensive assessment of their potential applications. This evaluation included sequencing analysis, genome classification, pH and temperature stability assessments, and in vitro bacterial inhibition assays. Further investigations involved analyzing histological and cytokine alterations in rats undergoing phage cocktail treatment for pneumonia. The therapeutic efficacy of the phages was validated, and transcriptomic studies of rat lung tissue during phage treatment revealed crucial changes in the immune system. The findings from our study underscore the potential of phages for future development as a treatment strategy and offer compelling evidence regarding immune system dynamics throughout the treatment process.IMPORTANCEDue to the growing problem of multi-drug-resistant bacteria, the use of phages is being considered as an alternative to antibiotics, and the genetic safety and application stability of phages determine the potential of phage application. The absence of drug resistance genes and virulence genes in the phage genome can ensure the safety of phage application, and the fact that phage can remain active in a wide range of temperatures and pH is also necessary for application. In addition, the effect evaluation of preclinical studies is especially important for clinical application. By simulating the immune response situation during the treatment process through mammalian models, the changes in animal immunity can be observed, and the effect of phage therapy can be further evaluated. Our study provides compelling evidence that phages hold promise for further development as therapeutic agents for Acinetobacter baumannii infections.
Asunto(s)
Infecciones por Acinetobacter , Acinetobacter baumannii , Bacteriófagos , Carbapenémicos , Modelos Animales de Enfermedad , Terapia de Fagos , Acinetobacter baumannii/virología , Acinetobacter baumannii/efectos de los fármacos , Animales , Infecciones por Acinetobacter/terapia , Infecciones por Acinetobacter/microbiología , Ratas , Terapia de Fagos/métodos , Carbapenémicos/farmacología , Bacteriófagos/fisiología , Bacteriófagos/genética , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Farmacorresistencia Bacteriana Múltiple , Masculino , Genoma Viral , Aguas Residuales , Neumonía/terapia , Neumonía/microbiología , Neumonía/virologíaRESUMEN
Phage therapy offers a promising approach to combat the growing threat of antimicrobial resistance. Yet, key questions remain regarding dosage, administration routes, combination therapy, and the causes of therapeutic failure. In this study, we focused on a novel lytic phage, ФAb4B, which specifically targeted the Acinetobacter baumannii strains with KL160 capsular polysaccharide, including the pan-drug resistant A. baumannii YQ4. ФAb4B exhibited the ability to effectively inhibit biofilm formation and eradicate mature biofilms independently of dosage. Additionally, it demonstrated a wide spectrum of antibiotic-phage synergy and did not show any cytotoxic or haemolytic effects. Continuous phage injections, both intraperitoneally and intravenously over 7 d, showed no acute toxicity in vivo. Importantly, phage therapy significantly improved neutrophil counts, outperforming ciprofloxacin. However, excessive phage injections suppressed neutrophil levels. The combinatorial treatment of phage-ciprofloxacin rescued 91% of the mice, a superior outcome compared to phage alone (67%). The efficacy of the combinatorial treatment was independent of phage dosage. Notably, prophylactic administration of the combinatorial regimen provided no protection, but even when combined with a delayed therapeutic regimen, it saved all the mice. Bacterial resistance to the phage was not a contributing factor to treatment failure. Our preclinical study systematically describes the lytic phage's effectiveness in both in vitro and in vivo settings, filling in crucial details about phage treatment against bacteriemia caused by A. baumannii, which will provide a robust foundation for the future of phage therapy.
Asunto(s)
Infecciones por Acinetobacter , Acinetobacter baumannii , Antibacterianos , Bacteriófagos , Biopelículas , Ciprofloxacina , Farmacorresistencia Bacteriana Múltiple , Terapia de Fagos , Acinetobacter baumannii/virología , Acinetobacter baumannii/efectos de los fármacos , Terapia de Fagos/métodos , Infecciones por Acinetobacter/terapia , Infecciones por Acinetobacter/microbiología , Animales , Biopelículas/efectos de los fármacos , Bacteriófagos/fisiología , Ratones , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Ciprofloxacina/uso terapéutico , Ciprofloxacina/farmacología , Modelos Animales de Enfermedad , Femenino , Ratones Endogámicos BALB CRESUMEN
Acinetobacter baumannii is one of the most important pathogens of healthcare-associated infections. The rising prevalence of multidrug-resistant A. baumannii (MRAB) strains and biofilm formation impact the outcome of conventional treatment. Phage-related therapy is a promising strategy to tame troublesome multidrug-resistant bacteria. Here, we isolated and evaluated a highly efficient lytic phage called MRABP9 from hospital sewage. The phage was a novel species within the genus Friunavirus and exhibited lytic activity against 2 other identified MRAB strains. Genomic analysis revealed it was a safe virulent phage and a pectate lyase domain was identified within its tail spike protein. MRABP9 showed potent bactericidal and anti-biofilm activity against MRAB, significantly delaying the time point of bacterial regrowth in vitro. Phage administration could rescue the mice from acute lethal MRAB infection. Considering its features, MRABP9 has the potential as an efficient candidate for prophylactic and therapeutic use against acute infections caused by MRAB strains.
Asunto(s)
Infecciones por Acinetobacter , Acinetobacter baumannii , Bacteriófagos , Farmacorresistencia Bacteriana Múltiple , Terapia de Fagos , Acinetobacter baumannii/virología , Acinetobacter baumannii/efectos de los fármacos , Animales , Infecciones por Acinetobacter/microbiología , Infecciones por Acinetobacter/terapia , Ratones , Bacteriófagos/genética , Bacteriófagos/fisiología , Terapia de Fagos/métodos , Genoma Viral , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Humanos , Femenino , Aguas del Alcantarillado/virologíaRESUMEN
Introduction . Acinetobacter baumannii is a critical priority pathogen for novel antimicrobials (World Health Organization) because of the rise in nosocomial infections and its ability to evolve resistance to last resort antibiotics. A. baumannii is thus a priority target for phage therapeutics. Two strains of a novel, virulent bacteriophage (LemonAid and Tonic) able to infect carbapenem-resistant A. baumannii (strain NCTC 13420), were isolated from environmental water samples collected through a citizen science programme.Gap statement. Phage-host coevolution can lead to emergence of host resistance, with a concomitant reduction in the virulence of host bacteria; a potential benefit to phage therapy applications.Methodology. In vitro and in vivo assays, genomics and microscopy techniques were used to characterize the phages; determine mechanisms and impact of phage resistance on host virulence, and the efficacy of the phages against A. baumannii.Results. A. baumannii developed resistance to both viruses, LemonAid and Tonic. Resistance came at a cost to virulence, with the resistant variants causing significantly reduced mortality in a Galleria mellonella larval in vivo model. A replicated 8 bp insertion increased in frequency (~40â% higher frequency than in the wild-type) within phage-resistant A. baumannii mutants, putatively resulting in early truncation of a protein of unknown function. Evidence from comparative genomics and an adsorption assay suggests this protein acts as a novel phage receptor site in A. baumannii. We find no evidence linking resistance to changes in capsule structure, a known virulence factor. LemonAid efficiently suppressed growth of A. baumanni in vitro across a wide range of titres. However, in vivo, while survival of A. baumannii infected larvae significantly increased with both remedial and prophylactic treatment with LemonAid (107 p.f.u. ml-1), the effect was weak and not sufficient to save larvae from morbidity and mortality.Conclusion. While LemonAid and Tonic did not prove effective as a treatment in a Galleria larvae model, there is potential to harness their ability to attenuate virulence in drug-resistant A. baumannii.
Asunto(s)
Infecciones por Acinetobacter , Acinetobacter baumannii , Bacteriófagos , Acinetobacter baumannii/virología , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/patogenicidad , Acinetobacter baumannii/genética , Bacteriófagos/genética , Bacteriófagos/fisiología , Virulencia , Infecciones por Acinetobacter/microbiología , Animales , Mariposas Nocturnas/microbiología , Mariposas Nocturnas/virología , Terapia de Fagos , Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Larva/microbiología , Larva/virologíaRESUMEN
Bacteriophages exert strong selection on their bacterial hosts to evolve resistance. At the same time, the fitness costs on bacteria following phage resistance may change their virulence, which may affect the therapeutic outcomes of phage therapy. In this study, we set out to assess the costs of phage resistance on the in vitro virulence of priority 1 nosocomial pathogenic bacterium, Acinetobacter baumannii. By subjecting phage-resistant variant Ev5-WHG of A. baumannii WHG40004 to several in vitro virulence profiles, we found that its resistance to phage is associated with reduced fitness in host microenvironments. Also, the mutant exhibited impaired adhesion and invasion to mammalian cells, as well as increased susceptibility to macrophage phagocytosis. Furthermore, the whole-genome sequencing of the mutant revealed that there exist multiple mutations which may play a role in phage resistance and altered virulence. Altogether, this study demonstrates that resistance to phage can significantly alter phenotypes associated with virulence in Acinetobacter baumannii.
Asunto(s)
Infecciones por Acinetobacter , Acinetobacter baumannii , Bacteriófagos , Fenotipo , Acinetobacter baumannii/virología , Acinetobacter baumannii/patogenicidad , Acinetobacter baumannii/genética , Virulencia/genética , Bacteriófagos/genética , Bacteriófagos/fisiología , Bacteriófagos/patogenicidad , Infecciones por Acinetobacter/microbiología , Animales , Humanos , Macrófagos/microbiología , Macrófagos/virología , Mutación , Fagocitosis , Secuenciación Completa del Genoma , RatonesRESUMEN
Carbapenem-resistant Acinetobacter baumannii (CRAB) has become a new threat in recent years, owing to its rapidly increasing resistance to antibiotics and new effective therapies are needed to combat this pathogen. Phage therapy is considered to be the most promising alternative for treating CRAB infections. In this study, a novel phage, Ab_WF01, which can lyse clinical CRAB, was isolated and characterized from hospital sewage. The multiplicity of infection, morphology, one-step growth curve, stability, sensitivity, and lytic activity of the phage were also investigated. The genome of phage Ab_WF01 was 41, 317 bp in size with a GC content of 39.12% and encoded 51 open reading frames (ORFs). tRNA, virulence, and antibiotic resistance genes were not detected in the phage genome. Comparative genomic and phylogenetic analyses suggest that phage Ab_WF01 is a novel species of the genus Friunavirus, subfamily Beijerinckvirinae, and family Autographiviridae. The in vivo results showed that phage Ab_WF01 significantly increased the survival rate of CRAB-infected Galleria mellonella (from 0% to 70% at 48 h) and mice (from 0% to 60% for 7 days). Moreover, after day 3 post-infection, phage Ab_WF01 reduced inflammatory response, with strongly ameliorated histological damage and bacterial clearance in infected tissue organs (lungs, liver, and spleen) in mouse CRAB infection model. Taken together, these results show that phage Ab_WF01 holds great promise as a potential alternative agent with excellent stability for against CRAB infections.
Asunto(s)
Infecciones por Acinetobacter , Acinetobacter baumannii , Bacteriófagos , Carbapenémicos , Genoma Viral , Terapia de Fagos , Filogenia , Aguas del Alcantarillado , Acinetobacter baumannii/virología , Acinetobacter baumannii/efectos de los fármacos , Aguas del Alcantarillado/virología , Aguas del Alcantarillado/microbiología , Animales , Carbapenémicos/farmacología , Bacteriófagos/genética , Bacteriófagos/fisiología , Bacteriófagos/clasificación , Bacteriófagos/aislamiento & purificación , Infecciones por Acinetobacter/microbiología , Ratones , Antibacterianos/farmacología , Sistemas de Lectura Abierta , Modelos Animales de Enfermedad , Mariposas Nocturnas/virología , Mariposas Nocturnas/microbiología , Composición de BaseRESUMEN
The genus Acinetobacter comprises both environmental and clinically relevant species associated with hospital-acquired infections. Among them, Acinetobacter baumannii is a critical priority bacterial pathogen, for which the research and development of new strategies for antimicrobial treatment are urgently needed. Acinetobacter spp. produce a variety of structurally diverse capsular polysaccharides (CPSs), which surround the bacterial cells with a thick protective layer. These surface structures are primary receptors for capsule-specific bacteriophages, that is, phages carrying tailspikes with CPS-depolymerizing/modifying activities. Phage tailspike proteins (TSPs) exhibit hydrolase, lyase, or esterase activities toward the corresponding CPSs of a certain structure. In this study, the data on all lytic capsule-specific phages infecting Acinetobacter spp. with genomes deposited in the NCBI GenBank database by January 2024 were summarized. Among the 149 identified TSPs encoded in the genomes of 143 phages, the capsular specificity (K specificity) of 46 proteins has been experimentally determined or predicted previously. The specificity of 63 TSPs toward CPSs, produced by various Acinetobacter K types, was predicted in this study using a bioinformatic analysis. A comprehensive phylogenetic analysis confirmed the prediction and revealed the possibility of the genetic exchange of gene regions corresponding to the CPS-recognizing/degrading parts of different TSPs between morphologically and taxonomically distant groups of capsule-specific Acinetobacter phages.
Asunto(s)
Acinetobacter , Cápsulas Bacterianas , Bacteriófagos , Genoma Viral , Filogenia , Bacteriófagos/genética , Bacteriófagos/enzimología , Bacteriófagos/clasificación , Acinetobacter/virología , Acinetobacter/genética , Acinetobacter/enzimología , Cápsulas Bacterianas/metabolismo , Cápsulas Bacterianas/genética , Proteínas de la Cola de los Virus/genética , Proteínas de la Cola de los Virus/metabolismo , Polisacáridos/metabolismo , Polisacáridos Bacterianos/metabolismo , Polisacáridos Bacterianos/genética , Acinetobacter baumannii/virología , Acinetobacter baumannii/genética , Acinetobacter baumannii/enzimología , Glicósido HidrolasasRESUMEN
Where there are bacteria, there will be bacteriophages. These viruses are known to be important players in shaping the wider microbial community in which they are embedded, with potential implications for human health. On the other hand, bacteria possess a range of distinct immune mechanisms that provide protection against bacteriophages, including the mutation or complete loss of the phage receptor, and CRISPR-Cas adaptive immunity. While our previous work showed how a microbial community may impact phage resistance evolution, little is known about the inverse, namely how interactions between phages and these different phage resistance mechanisms affect the wider microbial community in which they are embedded. Here, we conducted a 10-day, fully factorial evolution experiment to examine how phage impact the structure and dynamics of an artificial four-species bacterial community that includes either Pseudomonas aeruginosa wild-type or an isogenic mutant unable to evolve phage resistance through CRISPR-Cas. Additionally, we used mathematical modelling to explore the ecological interactions underlying full community behaviour, as well as to identify general principles governing the impacts of phage on community dynamics. Our results show that the microbial community structure is drastically altered by the addition of phage, with Acinetobacter baumannii becoming the dominant species and P. aeruginosa being driven nearly extinct, whereas P. aeruginosa outcompetes the other species in the absence of phage. Moreover, we find that a P. aeruginosa strain with the ability to evolve CRISPR-based resistance generally does better when in the presence of A. baumannii, but that this benefit is largely lost over time as phage is driven extinct. Finally, we show that pairwise data alone is insufficient when modelling our microbial community, both with and without phage, highlighting the importance of higher order interactions in governing multispecies dynamics in complex communities. Combined, our data clearly illustrate how phage targeting a dominant species allows for the competitive release of the strongest competitor while also contributing to community diversity maintenance and potentially preventing the reinvasion of the target species, and underline the importance of mapping community composition before therapeutically applying phage.
Asunto(s)
Bacteriófagos , Sistemas CRISPR-Cas , Microbiota , Pseudomonas aeruginosa , Bacteriófagos/fisiología , Bacteriófagos/genética , Pseudomonas aeruginosa/virología , Acinetobacter baumannii/virología , Mutación , Bacterias/virología , Bacterias/genéticaRESUMEN
OBJECTIVES: Acinetobacter Baumannii is an opportunistic nosocomial pathogen belonging to the Moraxellaceae family. The emergence of multidrug resistant strains of this pathogen caused many problems for hospitals and patients. The aim of the current study was to isolate, identify, and morphologically, physiologically, and in vivo analyze a new lytic bacteriophage targeting extensively drug-resistant (XDR) A. baumannii. MATERIALS AND METHODS: Different wastewater samples were tested for isolation of lytic bacteriophage against 19 A. baumannii isolates obtained from patients hospitalized in a hospital in Arak, Iran, from January 2019 to March 2019. The phenotypic and genotypic characteristics of A. baumannii strains (resistance genes including: adeA, adeB, adeC, adeR, adeS, ISAba1, blaOXA-23, blaOXA-24) were analyzed. The isolated phage characteristics including adsorption time, pH and thermal stability, host range, one-step growth rate, electron microscopy examination, and therapeutic efficacy of the phage were also investigated. Therapeutic efficacy of the phage was evaluated in a rat model with burn infection of XDR A. baumannii. The lesion image was taken on different days after burning and infection induction and was compared with phage untreated lesions. RESULTS: The results showed unique characteristics of the isolated phage (vB-AbauM-Arak1) including high specificity for Acinetobacter baumannii, stability at a relatively wide range of temperatures and pH values, short adsorption time, short latent period, and large burst size. In relation to the therapeutic efficacy of the phage, the lesion area decreased in phage-treated groups over 14 days than in those untreated, significantly (p < 0.05). CONCLUSION: Our findings demonstrated that isolated lytic phage was able to eliminate burn infections caused by XDR A. baumannii in a rat model. So, it may be recommended as alternative options toward to developing a treatment for extensively drug resistant Acinetobacter infections.
Asunto(s)
Infecciones por Acinetobacter , Acinetobacter baumannii , Bacteriófagos , Quemaduras , Farmacorresistencia Bacteriana Múltiple , Terapia de Fagos , Infecciones por Acinetobacter/microbiología , Infecciones por Acinetobacter/terapia , Infecciones por Acinetobacter/virología , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/virología , Animales , Antibacterianos/farmacología , Bacteriófagos/genética , Bacteriófagos/aislamiento & purificación , Quemaduras/microbiología , Quemaduras/terapia , Quemaduras/virología , Modelos Animales de Enfermedad , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Irán , RatasRESUMEN
Multidrug-resistant Acinetobacter baumannii (MDR A. baumannii) is one of the ESKAPE pathogens that restricts available treatment options. MDR A. baumannii is responsible for a dramatic increase in case numbers of a wide variety of infections, including skin and soft tissue infections (SSTIs), resulting in pyoderma, surgical debridement, and necrotizing fasciitis. To investigate an alternative medical treatment for SSTIs, a broad range lytic Acinetobacter phage, vB _AbP_ABWU2101 (phage vABWU2101), for lysing MDR A. baumannii in associated SSTIs was isolated and the biological aspects of this phage were investigated. Morphological characterization and genomic analysis revealed that phage vABWU2101 was a new species in the Friunavirus, Beijerinckvirinae, family Autographiviridae, and order Caudovirales. Antibiofilm activity of phage vABWU2101 demonstrated good activity against both preformed biofilms and biofilm formation. The combination of phage vABWU2101 and tigecycline showed synergistic antimicrobial activities against planktonic and biofilm cells. Scanning electron microscopy confirmed that the antibacterial efficacy of the combination of phage vABWU2101 and tigecycline was more effective than the phage or antibiotic alone. Hence, our findings could potentially be used to develop a therapeutic option for the treatment of SSTIs caused by MDR A. baumannii.
Asunto(s)
Infecciones por Acinetobacter/terapia , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/virología , Antibacterianos/farmacología , Bacteriófagos/fisiología , Enfermedades de la Piel/terapia , Infecciones de los Tejidos Blandos/terapia , Tigeciclina/farmacología , Infecciones por Acinetobacter/tratamiento farmacológico , Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/genética , Acinetobacter baumannii/fisiología , Bacteriófagos/clasificación , Bacteriófagos/genética , Bacteriófagos/aislamiento & purificación , Biopelículas/efectos de los fármacos , Terapia Combinada , Farmacorresistencia Bacteriana Múltiple , Genoma Viral , Humanos , Filogenia , Enfermedades de la Piel/tratamiento farmacológico , Enfermedades de la Piel/microbiología , Infecciones de los Tejidos Blandos/tratamiento farmacológico , Infecciones de los Tejidos Blandos/microbiologíaRESUMEN
The emergence of carbapenem-resistant Acinetobacter baumannii (CRAB) has been increasingly reported, leading to greater challenges in treating infections. With the development of phage therapy and phage-antibiotic combinations, it is promising to improve the treatment of bacterial infections. In the present study, a novel vB_AbaP_WU2001 (vWU2001) phage-specific CRAB with a genome of 40,792 bp was isolated. Genomic analysis disclosed that it belongs to the Autographiviridae family of the order Caudovirales. Phage vWU2001 had a broad host range with a high adsorption rate, short latent period, large burst size and good stability. The phage could reduce preformed biofilms and inhibit biofilm formation. The combination of phage vWU2001 and colistin had significantly higher bacterial growth inhibition activity than that of phage, or colistin alone. The efficacy of the combined treatment was also evaluated in Galleria mellonella. Evaluation of its therapeutic potential showed that the combination of phage and colistin resulted in a significantly greater increase in G. mellonella survival and in bacterial clearance, as compared with that of phage or colistin alone, indicating that the combination was synergistic against CRAB. The results demonstrated that phage vWU2001 has the potential to be developed as an antibacterial agent.
Asunto(s)
Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/virología , Bacteriófagos , Carbapenémicos/farmacología , Colistina/farmacología , Podoviridae , Infecciones por Acinetobacter/microbiología , Infecciones por Acinetobacter/terapia , Biopelículas , Farmacorresistencia Bacteriana , Sinergismo Farmacológico , Terapia de Fagos , Podoviridae/genéticaRESUMEN
Acinetobacter baumannii has emerged as an important opportunistic pathogen worldwide, being responsible for large outbreaks for nosocomial infections, primarily in intensive care units. A. baumannii ATCC 19606T is the species type strain, and a reference organism in many laboratories due to its low virulence, amenability to genetic manipulation and extensive antibiotic susceptibility. We wondered if frequent propagation of A. baumannii ATCC 19606T in different laboratories may have driven micro- and macro-evolutionary events that could determine inter-laboratory differences of genome-based data. By combining Illumina MiSeq, MinION and Sanger technologies, we generated a high-quality whole-genome sequence of A. baumannii ATCC 19606T, then performed a comparative genome analysis between A. baumannii ATCC 19606T strains from several research laboratories and a reference collection. Differences between publicly available ATCC 19606T genome sequences were observed, including SNPs, macro- and micro-deletions, and the uneven presence of a 52 kb prophage belonging to genus Vieuvirus. Two plasmids, pMAC and p1ATCC19606, were invariably detected in all tested strains. The presence of a putative replicase, a replication origin containing four 22-mer direct repeats, and a toxin-antitoxin system implicated in plasmid stability were predicted by in silico analysis of p1ATCC19606, and experimentally confirmed. This work refines the sequence, structure and functional annotation of the A. baumannii ATCC 19606T genome, and highlights some remarkable differences between domesticated strains, likely resulting from genetic drift.
Asunto(s)
Acinetobacter baumannii/clasificación , Infección Hospitalaria/microbiología , Variación Genética , Secuenciación Completa del Genoma/métodos , Acinetobacter baumannii/genética , Acinetobacter baumannii/aislamiento & purificación , Acinetobacter baumannii/virología , Evolución Molecular , Genoma Bacteriano , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Unidades de Cuidados Intensivos , Plásmidos/genética , Polimorfismo de Nucleótido Simple , Profagos/genética , Profagos/aislamiento & purificación , Eliminación de SecuenciaRESUMEN
Multidrug-resistant bacterial infections are becoming increasingly common, with only few last-resort antibiotics such as colistin available for clinical therapy. An alternative therapeutic strategy gaining momentum is phage therapy, which has the advantage of not being affected by bacterial resistance to antibiotics. However, a major challenge in phage therapy is the rapid emergence of phage-resistant bacteria. In this work, our main aim was to understand the mechanisms of phage-resistance used by the top priority pathogen Acinetobacter baumannii. We isolated the novel phage Phab24, capable of infecting colistin-sensitive and -resistant strains of A. baumannii. After co-incubating Phab24 with its hosts, we obtained phage-resistant mutants which were characterized on both genotypic and phenotypic levels. Using whole genome sequencing, we identified phage-resistant strains that displayed mutations in genes that alter the architecture of the bacterial envelope at two levels: the capsule and the outer membrane. Using an adsorption assay, we confirmed that phage Phab24 uses the bacterial capsule as its primary receptor, with the outer membrane possibly serving as the secondary receptor. Interestingly, the phage-resistant isolates were less virulent compared to the parental strains in a Galleria mellonella infection model. Most importantly, we observed that phage-resistant bacteria that evolved in the absence of antibiotics exhibited an increased sensitivity to colistin, even though the antibiotic resistance mechanism per se remained unaltered. This increase in antibiotic sensitivity is a direct consequence of the phage-resistance mechanism, and could potentially be exploited in the clinical setting.
Asunto(s)
Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/virología , Antibacterianos/farmacología , Bacteriófagos/fisiología , Colistina/farmacología , Infecciones por Acinetobacter/tratamiento farmacológico , Infecciones por Acinetobacter/terapia , Acinetobacter baumannii/genética , Acinetobacter baumannii/fisiología , Bacteriófagos/genética , Bacteriófagos/aislamiento & purificación , Farmacorresistencia Bacteriana , Humanos , Pruebas de Sensibilidad Microbiana , Secuenciación Completa del GenomaRESUMEN
Drug resistance of methicillin-resistant Staphylococcus aureus, extended-spectrum beta-lactamases-producing Escherichia coli and Klebsiella pneumoniae and multidrug-resistant Acinetobacter baumannii are also cited as one of the most important causes of community and hospital acquired infections. Phage therapy can be used as a therapeutic method for the treatment of infections caused by these bacteria. The aim of this study was to isolate bacteriophages from municipal wastewater and assess their effects against drug resistant bacterial strains. The single agar layer technique was used to investigate the bacteriolytic effect of bacteriophages. Then, the double agar layer technique was used to observe phage plaques and the transmission electron microscopy was used to study the morphology of the bacteriophages. Transparent plaque formation in a double agar layer test of methicillin-resistant S. aureus and extended-spectrum beta-lactamases-producing E. coli and K. pneumoniae indicated the lysis of bacterial cells by isolated bacteriophages. No bacteriophage against A. baumannii was isolated from municipal wastewater. The morphology of these bacteriophages was also identified by electron microscopy. The results of this study showed that bacteriophages act specifically and due to the increasing level of antibiotic resistance, phage therapy as a new treatment can open a new horizon for the treatment of multidrug resistant bacteria.
Asunto(s)
Bacterias , Bacteriófagos , Acinetobacter baumannii/virología , Antibacterianos/farmacología , Bacterias/virología , Bacteriófagos/aislamiento & purificación , Bacteriófagos/fisiología , Escherichia coli/virología , Klebsiella pneumoniae/virología , Staphylococcus aureus Resistente a Meticilina/virología , Pruebas de Sensibilidad Microbiana , Aguas Residuales/microbiologíaRESUMEN
Acinetobacter baumannii appears to be one of the most crucial nosocomial pathogens. A possible component of antimicrobial therapy for infections caused by extremely drug-resistant A. baumannii strains may be specific lytic bacteriophages or phage-derived enzymes. In the present study, we observe the biological features, genomic organization, and phage-host interaction strategy of novel virulent bacteriophage Aristophanes isolated on A. baumannii strain having K26 capsular polysaccharide structure. According to phylogenetic analysis phage Aristophanes can be classified as a representative of a new distinct genus of the subfamily Beijerinckvirinae of the family Autographiviridae. This is the first reported A. baumannii phage carrying tailspike deacetylase, which caused O-acetylation of one of the K26 sugar residues.