RESUMO
The association of Helicobacter pylori to several gastric diseases, such as chronic gastritis, peptic ulcer disease, and gastric cancer, and its high prevalence worldwide, raised the necessity to use methods for a proper and fast diagnosis and monitoring the pathogen eradication. Available diagnostic methods can be classified as invasive or non-invasive, and the selection of the best relies on the clinical condition of the patient, as well as on the sensitivity, specificity, and accessibility of the diagnostic test. This review summarises all diagnostic methods currently available, including the invasive methods: endoscopy, histology, culture, and molecular methods, and the rapid urease test (RUT), as well as the non-invasive methods urea breath test (UBT), serological assays, biosensors, and microfluidic devices and the stool antigen test (SAT). Moreover, it lists the diagnostic advantages and limitations, as well as the main advances for each methodology. In the end, research on the development of new diagnostic methods, such as bacteriophage-based H. pylori diagnostic tools, is also discussed.
Assuntos
Infecções por Helicobacter , Helicobacter pylori , Humanos , Helicobacter pylori/genética , Sensibilidade e Especificidade , Infecções por Helicobacter/diagnóstico , Urease , FezesRESUMO
Staphylococcus aureus is one of the most relevant mastitis pathogens in dairy cattle, and the acquisition of antimicrobial resistance genes presents a significant health issue in both veterinary and human fields. Among the different strategies to tackle S. aureus infection in livestock, bacteriophages have been thoroughly investigated in the last decades; however, few specimens of the so-called jumbo phages capable of infecting S. aureus have been described. Herein, we report the biological, genomic, and structural proteomic features of the jumbo phage vB_SauM-UFV_DC4 (DC4). DC4 exhibited a remarkable killing activity against S. aureus isolated from the veterinary environment and stability at alkaline conditions (pH 4 to 12). The complete genome of DC4 is 263,185 bp (GC content: 25%), encodes 263 predicted CDSs (80% without an assigned function), 1 tRNA (Phe-tRNA), multisubunit RNA polymerase, and an RNA-dependent DNA polymerase. Moreover, comparative analysis revealed that DC4 can be considered a new viral species belonging to a new genus DC4 and showed a similar set of lytic proteins and depolymerase activity with closely related jumbo phages. The characterization of a new S. aureus jumbo phage increases our understanding of the diversity of this group and provides insights into the biotechnological potential of these viruses. KEY POINTS: ⢠vB_SauM-UFV_DC4 is a new viral species belonging to a new genus within the class Caudoviricetes. ⢠vB_SauM-UFV_DC4 carries a set of RNA polymerase subunits and an RNA-directed DNA polymerase. ⢠vB_SauM-UFV_DC4 and closely related jumbo phages showed a similar set of lytic proteins.
Assuntos
Bacteriófagos , Fagos de Staphylococcus , Animais , Bovinos , Feminino , Humanos , Fagos de Staphylococcus/genética , Staphylococcus aureus/genética , Proteômica , Genoma Viral , Genômica , Bacteriófagos/genética , RNA Polimerases Dirigidas por DNA/genética , RNA de TransferênciaRESUMO
Salmonella Enteritidis is the main serotype responsible for human salmonellosis in the European Union. One of the main sources of Salmonella spp. in the food chain are poultry products, such as eggs or chicken meat. In recent years, molecular methods have become an alternative to culture dependent methods for the rapid screening of Salmonella spp. In this work, the strain S. Enteritidis S1400, and previously isolated and characterized bacteriophage PVP-SE2, were used to develop and evaluate a same-day detection method combining Phage Amplification and Loop-mediated isothermal amplification (PA-LAMP) to specifically detect viable S. Enteritidis in chicken breast. This method is based on the detection of the phage DNA rather than bacterial DNA. The virus is added to the sample during pre-enrichment in buffered peptone water, where it replicates in the presence of viable S. Enteritidis. The detection of phage DNA allows, on the one hand to detect viable bacteria, since viruses only replicate in them, and on the other hand to increase the sensitivity of the method since for each infected S. Enteritidis cell, hundreds of new viruses are produced. Two different PA-LAMP detection strategies were evaluated, a real time fluorescence and a naked-eye detection. The present method could down to 0.2 fg/µL of pure phage DNA and a concentration of viral particles of 2.2 log PFU/mL. After a short Salmonella recovery step of 3 h and a co-culture of 4 h of the samples with phage particles, both real-time fluorescence and naked-eye method showed a LoD95 of 6.6 CFU/25 g and a LoD50 of 1.5/25 g in spiked chicken breast samples. The entire detection process, including DNA extraction and LAMP analysis, can be completed in around 8 h. In the current proof-of-concept, the novel PA-LAMP obtained comparable results to those of the reference method ISO 6579, to detect Salmonella Enteritidis in poultry meat.
Assuntos
Bacteriófagos , Salmonella enteritidis , Animais , Humanos , Salmonella enteritidis/genética , Aves Domésticas , Microbiologia de Alimentos , Carne/microbiologiaRESUMO
Antimicrobial resistance (AMR) is considered one of the greatest threats to global health. Methicillin-resistant Staphylococcus aureus (MRSA) remains at the core of this threat, accounting for about 90% of S. aureus infections widespread in the community and hospital settings. In recent years, the use of nanoparticles (NPs) has emerged as a promising strategy to treat MRSA infections. NPs can act directly as antibacterial agents via antibiotic-independent activity and/or serve as drug delivery systems (DDSs), releasing loaded antibiotics. Nonetheless, directing NPs to the infection site is fundamental for effective MRSA treatment so that highly concentrated therapeutic agents are delivered to the infection site while directly reducing the toxicity to healthy human cells. This leads to decreased AMR emergence and less disturbance of the individual's healthy microbiota. Hence, this review compiles and discusses the scientific evidence related to targeted NPs developed for MRSA treatment.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Nanopartículas , Infecções Estafilocócicas , Humanos , Staphylococcus aureus , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Sistemas de Liberação de Medicamentos , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologiaRESUMO
Acinetobacter baumannii is an important pathogen causative of health care-associated infections and is able to rapidly develop resistance to all known antibiotics, including colistin. As an alternative therapeutic agent, we have isolated a novel myovirus (vB_AbaM_B9) which specifically infects and makes lysis from without in strains of the K45 and K30 capsule types, respectively. Phage B9 has a genome of 93,641 bp and encodes 167 predicted proteins, of which 29 were identified by mass spectrometry. This phage holds a capsule depolymerase (B9gp69) able to digest extracted exopolysaccharides of both K30 and K45 strains and remains active in a wide range of pH values (5 to 9), ionic strengths (0 to 500 mM), and temperatures (20 to 80°C). B9gp69 was demonstrated to be nontoxic in a cell line model of the human lung and to make the K45 strain fully susceptible to serum killing in vitro Contrary to the case with phage, no resistance development was observed by bacteria targeted with the B9gp69. Therefore, capsular depolymerases may represent attractive antimicrobial agents against A. baumannii infections.IMPORTANCE Currently, phage therapy has revived interest for controlling hard-to-treat bacterial infections. Acinetobacter baumannii is an emerging Gram-negative pathogen able to cause a variety of nosocomial infections. Additionally, this species is becoming more resistant to several classes of antibiotics. Here we describe the isolation of a novel lytic myophage B9 and its recombinant depolymerase. While the phage can be a promising alternative antibacterial agent, its success in the market will ultimately depend on new regulatory frameworks and general public acceptance. We therefore characterized the phage-encoded depolymerase, which is a natural enzyme that can be more easily managed and used. To our knowledge, the therapeutic potential of phage depolymerase against A. baumannii is still unknown. We show for the first time that the K45 capsule type is an important virulence factor of A. baumannii and that capsule removal via the recombinant depolymerase activity helps the host immune system to combat the bacterial infection.
Assuntos
Glicosídeo Hidrolases/metabolismo , Myoviridae/genética , Myoviridae/metabolismo , Acinetobacter baumannii/virologia , Cápsulas Bacterianas/fisiologia , Cápsulas Bacterianas/virologia , Bacteriófagos/genética , DNA Viral/genética , Genoma Viral , Glicosídeo Hidrolases/genética , Humanos , Fases de Leitura Aberta/genética , Análise de Sequência de DNA/métodos , Proteínas Virais/metabolismoRESUMO
Healthcare-associated infections (HCAIs) affect hundreds of millions of patients, representing a significant burden for public health. They are usually associated to multidrug resistant bacteria, which increases their incidence and severity. Bloodstream infections are among the most frequent and life-threatening HCAIs, with Enterococcus and Staphylococcus among the most common isolated pathogens. The correct and fast identification of the etiological agents is crucial for clinical decision-making, allowing to rapidly select the appropriate antimicrobial and to prevent from overuse and misuse of antibiotics and the consequent increase in antimicrobial resistance. Conventional culture methods are still the gold standard to identify these pathogens, however, are time-consuming and may lead to erroneous diagnosis, which compromises an efficient treatment. (Bacterio)phage receptor binding proteins (RBPs) are the structures responsible for the high specificity conferred to phages against bacteria and thus are very attractive biorecognition elements with high potential for specific detection and identification of pathogens. Taking into account all these facts, we have designed and developed a new, fast, accurate, reliable and unskilled diagnostic method based on newly identified phage RBPs and spectrofluorometric techniques that allows the multiplex detection of Enterococcus and Staphylococcus in blood samples in less than 1.5 hr after an enrichment step.
Assuntos
Bacteriemia , Bacteriófagos/genética , Enterococcus , Proteínas Recombinantes de Fusão , Staphylococcus , Proteínas Virais , Animais , Bacteriemia/sangue , Bacteriemia/diagnóstico , Receptores de Bacteriófagos/química , Receptores de Bacteriófagos/metabolismo , Enterococcus/química , Enterococcus/metabolismo , Cavalos , Limite de Detecção , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Staphylococcus/química , Staphylococcus/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Phages are recognized as the most abundant and diverse entities on the planet. Their diversity is determined predominantly by their dynamic adaptation capacities when confronted with different selective pressures in an endless cycle of coevolution with a widespread group of bacterial hosts. At the end of the infection cycle, progeny virions are confronted with a rigid cell wall that hinders their release into the environment and the opportunity to start a new infection cycle. Consequently, phages encode hydrolytic enzymes, called endolysins, to digest the peptidoglycan. In this work, we bring to light all phage endolysins found in completely sequenced double-stranded nucleic acid phage genomes and uncover clues that explain the phage-endolysin-host ecology that led phages to recruit unique and specialized endolysins.
Assuntos
Bacteriófagos/enzimologia , Endopeptidases/genética , Endopeptidases/metabolismo , Biologia Computacional , Hidrólise , Peptidoglicano/metabolismo , Filogenia , Homologia de Sequência de Aminoácidos , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
(Bacterio)phage PVP-SE1, isolated from a German wastewater plant, presents a high potential value as a biocontrol agent and as a diagnostic tool, even compared to the well-studied typing phage Felix 01, due to its broad lytic spectrum against different Salmonella strains. Sequence analysis of its genome (145,964 bp) shows it to be terminally redundant and circularly permuted. Its G+C content, 45.6 mol%, is lower than that of its hosts (50 to 54 mol%). We found a total of 244 open reading frames (ORFs), representing 91.6% of the coding capacity of the genome. Approximately 46% of encoded proteins are unique to this phage, and 22.1% of the proteins could be functionally assigned. This myovirus encodes a large number of tRNAs (n=24), reflecting its lytic capacity and evolution through different hosts. Tandem mass spectrometric analysis using electron spray ionization revealed 25 structural proteins as part of the mature phage particle. The genome sequence was found to share homology with 140 proteins of the Escherichia coli bacteriophage rV5. Both phages are unrelated to any other known virus, which suggests that an "rV5-like virus" genus should be created within the Myoviridae to contain these two phages.
Assuntos
DNA Viral/genética , Genoma Viral , Myoviridae/química , Myoviridae/genética , Fagos de Salmonella/química , Fagos de Salmonella/genética , Proteínas Virais/análise , Composição de Bases , Colífagos/genética , DNA Viral/química , Alemanha , Especificidade de Hospedeiro , Dados de Sequência Molecular , Myoviridae/classificação , Myoviridae/fisiologia , Fases de Leitura Aberta , Proteoma/análise , Salmonella/virologia , Fagos de Salmonella/classificação , Fagos de Salmonella/fisiologia , Análise de Sequência de DNA , Homologia de Sequência , Espectrometria de Massas em Tandem , Microbiologia da ÁguaRESUMO
BACKGROUND: Campylobacter is the leading cause of foodborne diseases worldwide. Bacteriophages (phages) are naturally occurring predators of bacteria, ubiquitous in the environment, with high host specificity and thus considered an appealing option to control bacterial pathogens. Nevertheless for an effective use of phages as antimicrobial agents, it is important to understand phage biology which renders crucial the analysis of phage genomes and proteomes. The lack of sequence data from Campylobacter phages adds further importance to these studies. METHODS: vB_CcoM-IBB_35 is a broad lytic spectrum Myoviridae Campylobacter phage with high potential for therapeutic use. The genome of this phage was obtained by pyrosequencing and the sequence data was further analyzed. The proteomic analysis was performed by SDS-PAGE and Mass spectrometry. RESULTS AND CONCLUSIONS: The DNA sequence data of vB_CcoM-IBB_35 consists of five contigs for a total of 172,065 bp with an average GC content of 27%. Attempts to close the gaps between contigs were unsuccessful since the DNA preparations appear to contain substances that inhibited Taq and Ï29 polymerases. From the 210 identified ORFs, around 60% represent proteins that were not functionally assigned. Homology exists with members of the Teequatrovirinae namely for T4 proteins involved in morphogenesis, nucleotide metabolism, transcription, DNA replication and recombination. Tandem mass spectrometric analysis revealed 38 structural proteins as part of the mature phage particle. CONCLUSIONS: Genes encoding proteins involved in the carbohydrate metabolism along with several incidences of gene duplications, split genes with inteins and introns have been rarely found in other phage genomes yet are found in this phage. We identified the genes encoding for tail fibres and for the lytic cassette, this later, expressing enzymes for bacterial capsular polysaccharides (CPS) degradation, which has not been reported before for Campylobacter phages.
Assuntos
Bacteriófagos/química , Bacteriófagos/genética , Campylobacter coli/virologia , Genoma Viral , Proteoma , DNA Viral/química , DNA Viral/genética , Eletroforese em Gel de Poliacrilamida , Ordem dos Genes , Genes Virais , Dados de Sequência Molecular , Myoviridae/química , Myoviridae/genética , Análise de Sequência de DNA , Espectrometria de Massas em Tandem , Proteínas Virais/química , Proteínas Virais/isolamento & purificaçãoRESUMO
Bacterial pathogens are progressively adapting to current antimicrobial therapies with severe consequences for patients and global health care systems. This is critically underscored by the rise of methicillin resistant Staphylococcus aureus (MRSA) and other biofilm-forming staphylococci. Accordingly, alternative strategies have been explored to fight such highly multidrug resistant microorganisms, including antimicrobial photodynamic therapy (aPDT) and phage therapy. aPDT has the great advantage that it does not elicit resistance, while phage therapy allows targeting of specific pathogens. In the present study, we aimed to merge these benefits by conjugating the cell-binding domain (CBD3) of a Staphylococcus aureus phage endolysin to a photoactivatable silicon phthalocyanine (IRDye 700DX) for the development of a Staphylococcus-targeted aPDT approach. We show that, upon red-light activation, the resulting CBD3-700DX conjugate generates reactive oxygen species that effectively kill high loads of planktonic and biofilm-resident staphylococci, including MRSA. Furthermore, CBD3-700DX is readily internalized by mammalian cells, where it allows the targeted killing of intracellular MRSA upon photoactivation. Intriguingly, aPDT with CBD3-700DX also affects mammalian cells with internalized MRSA, but it has no detectable side effects on uninfected cells. Altogether, we conclude that CBD3 represents an attractive targeting agent for Staphylococcus-specific aPDT, irrespective of planktonic, biofilm-embedded, or intracellular states of the bacterium. IMPORTANCE Antimicrobial resistance is among the biggest threats to mankind today. There are two alternative antimicrobial therapies that may help to control multidrug-resistant bacteria. In phage therapy, natural antagonists of bacteria, lytic phages, are harnessed to fight pathogens. In antimicrobial photodynamic therapy (aPDT), a photosensitizer, molecular oxygen, and light are used to produce reactive oxygen species (ROS) that inflict lethal damage on pathogens. Since aPDT destroys multiple essential components in targeted pathogens, aPDT resistance is unlikely. However, the challenge in aPDT is to maximize target specificity and minimize collateral oxidative damage to host cells. We now present an antimicrobial approach that combines the best features of both alternative therapies, namely, the high target specificity of phages and the efficacy of aPDT. This is achieved by conjugating the specific cell-binding domain from a phage protein to a near-infrared photosensitizer. aPDT with the resulting conjugate shows high target specificity toward MRSA with minimal side effects.
Assuntos
Antibacterianos/farmacologia , Endopeptidases/farmacologia , Fotoquimioterapia , Infecções Estafilocócicas/microbiologia , Fagos de Staphylococcus/química , Staphylococcus/efeitos dos fármacos , Staphylococcus/fisiologia , Animais , Antibacterianos/química , Biofilmes/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla , Endopeptidases/química , Endopeptidases/metabolismo , Humanos , Indóis/química , Luz , Compostos de Organossilício/química , Fármacos Fotossensibilizantes/química , Espécies Reativas de Oxigênio/metabolismo , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus/virologia , Fagos de Staphylococcus/metabolismoRESUMO
Klebsiella pneumoniae is a Gram-negative bacterium that has become one of the leading causes of life-threatening healthcare-associated infections (HAIs), including pneumonia and sepsis. Moreover, due to its increasingly antibiotic resistance, K. pneumoniae has been declared a global top priority concern. The problem of K. pneumoniae infections is due, in part, to the inability to detect this pathogen rapidly and accurately and thus to treat patients within the early stages of infections. The success in bacterial detection is greatly dictated by the biorecognition molecule used, with the current diagnostic tools relying on expensive probes often lacking specificity and/or sensitivity. (Bacterio)phage receptor-binding proteins (RBPs) are responsible for the recognition and adsorption of phages to specific bacterial host receptors and thus present high potential as biorecognition molecules. In this study, we report the identification and characterization of a novel RBP from the K. pneumoniae phage KpnM6E1 that presents high specificity against the target bacteria and high sensitivity (80%) to recognize K. pneumoniae strains. Moreover, adsorption studies validated the role of gp86 in the attachment to bacterial receptors, as it highly inhibits (86%) phage adsorption to its Klebsiella host. Overall, in this study, we unravel the role and potential of a novel Klebsiella phage RBP as a powerful tool to be used coupled with analytical techniques or biosensing platforms for the diagnosis of K. pneumoniae infections.
Assuntos
Receptores de Bacteriófagos , Infecções por Klebsiella , Proteínas de Transporte , Humanos , Klebsiella , Klebsiella pneumoniaeRESUMO
Acinetobacter pittii is a species that belong to the Acinetobacter calcoaceticus-baumannii complex, increasingly recognized as major nosocomial bacterial pathogens, often associated with multiple drug-resistances. The capsule surrounding the bacteria represents a main virulence factor, helping cells avoid phage predation and host immunity. Accordingly, a better understanding of the phage infection mechanisms is required to efficiently develop phage therapy against Acinetobacter of different capsular types. Here, we report the isolation of the novel A. pittii-infecting Fri1-like phage vB_Api_3043-K38 (3043-K38) of the Podoviridae morphotype, from sewage samples. Its 41,580 bp linear double-stranded DNA genome harbours 53 open reading frames and 302 bp of terminal repeats. We show that all studied Acinetobacter Fri1-like viruses have highly similar genomes, which differentiate only at the genes coding for tailspike, likely to adapt to different host receptors. The isolated phage 3043-K38 specifically recognizes an untapped Acinetobacter K38 capsule type via a novel tailspike with K38 depolymerase activity. The recombinant K38 depolymerase region of the tailspike (center-end region) forms a thermostable trimer, and quickly degrades capsules. When the K38 depolymerase is applied to the cells, it makes them resistant to phage predation. Interestingly, while K38 depolymerase treatments do not synergize with antibiotics, it makes bacterial cells highly susceptible to the host serum complement. In summary, we characterized a novel phage-encoded K38 depolymerase, which not only advances our understanding of phage-host interactions, but could also be further explored as a new antibacterial agent against drug-resistant Acinetobacter.
RESUMO
Bacteriophages (phages) are ubiquitous entities present in every conceivable habitat as a result of their bacterial parasitism. Their prevalence and impact in the ecology of bacterial communities and their ability to control pathogens make their characterization essential, particularly of new phages, improving knowledge and potential application. The isolation and characterization of a new lytic phage against Sphaerotilus natans strain DSM 6575, named vB_SnaP-R1 (SnaR1), is here described. Besides being the first sequenced genome of a Sphaerotilus natans infecting phage, 99% of its 41507 bp genome lacks homology with any other sequenced phage, revealing its uniqueness and previous lack of knowledge. Moreover, SnaR1 is the first Podoviridae phage described infecting this bacterium. Sphaerotilus natans is an important filamentous bacterium due to its deleterious effect on wastewater treatment plants (WWTP) and thus, phages may play a role as novel biotechnological tools against filamentous overgrowth in WWTP. The lytic spectrum of SnaR1 was restricted to its host strain, infecting only one out of three S. natans strains and infection assays revealed its ability to reduce bacterial loads. Results suggest SnaR1 as the prototype of a new phage genus and demonstrates its potential as a non-chemical alternative to reduce S. natans DSM 6575 cells.
Assuntos
Bacteriófagos , Sphaerotilus , Purificação da Água , Bacteriófagos/genética , Sequência de Bases , Genoma Viral , Sphaerotilus/genéticaRESUMO
Although different strategies to control biofilm formation on endotracheal tubes have been proposed, there are scarce scientific data on applying phages for both removing and preventing Pseudomonas aeruginosa biofilms on the device surface. Here, the anti-biofilm capacity of five bacteriophages was evaluated by a high content screening assay. We observed that biofilms were significantly reduced after phage treatment, especially in multidrug-resistant strains. Considering the anti-biofilm screens, two phages were selected as cocktail components, and the cocktail's ability to prevent colonization of the endotracheal tube surface was tested in a dynamic biofilm model. Phage-coated tubes were challenged with different P. aeruginosa strains. The biofilm growth was monitored from 24 to 168 h by colony forming unit counting, metabolic activity assessment, and biofilm morphology observation. The phage cocktail promoted differences of bacterial colonization; nonetheless, the action was strain dependent. Phage cocktail coating did not promote substantial changes in metabolic activity. Scanning electron microscopy revealed a higher concentration of biofilm cells in control, while tower-like structures could be observed on phage cocktail-coated tubes. These results demonstrate that with the development of new coating strategies, phage therapy has potential in controlling the endotracheal tube-associated biofilm.
RESUMO
BACKGROUND: Poultry meat is one of the most important sources of human campylobacteriosis, an acute bacterial enteritis which is a major problem worldwide. Campylobacter coli and Campylobacter jejuni are the most common Campylobacter species associated with this disease. These pathogens live in the intestinal tract of most avian species and under commercial conditions they spread rapidly to infect a high proportion of the flock, which makes their treatment and prevention very difficult. Bacteriophages (phages) are naturally occurring predators of bacteria with high specificity and also the capacity to evolve to overcome bacterial resistance. Therefore phage therapy is a promising alternative to antibiotics in animal production. This study tested the efficacy of a phage cocktail composed of three phages for the control of poultry infected with C. coli and C. jejuni. Moreover, it evaluated the effectiveness of two routes of phage administration (by oral gavage and in feed) in order to provide additional information regarding their future use in a poultry unit. RESULTS: The results indicate that experimental colonisation of chicks was successful and that the birds showed no signs of disease even at the highest dose of Campylobacter administered. The phage cocktail was able to reduce the titre of both C. coli and C. jejuni in faeces by approximately 2 log10 cfu/g when administered by oral gavage and in feed. This reduction persisted throughout the experimental period and neither pathogen regained their former numbers. The reduction in Campylobacter titre was achieved earlier (2 days post-phage administration) when the phage cocktail was incorporated in the birds' feed. Campylobacter strains resistant to phage infection were recovered from phage-treated chickens at a frequency of 13%. These resistant phenotypes did not exhibit a reduced ability to colonize the chicken guts and did not revert to sensitive types. CONCLUSIONS: Our findings provide further evidence of the efficacy of phage therapy for the control of Campylobacter in poultry. The broad host range of the novel phage cocktail enabled it to target both C. jejuni and C. coli strains. Moreover the reduction of Campylobacter by approximately 2 log10cfu/g, as occurred in our study, could lead to a 30-fold reduction in the incidence of campylobacteriosis associated with consumption of chicken meals (according to mathematical models). To our knowledge this is the first report of phage being administered in feed to Campylobacter-infected chicks and our results show that it lead to an earlier and more sustainable reduction of Campylobacter than administration by oral gavage. Therefore the present study is of extreme importance as it has shown that administering phages to poultry via the food could be successful on a commercial scale.
Assuntos
Bacteriófagos/fisiologia , Infecções por Campylobacter/veterinária , Campylobacter coli/virologia , Campylobacter jejuni/virologia , Galinhas , Doenças das Aves Domésticas/prevenção & controle , Animais , Infecções por Campylobacter/microbiologia , Infecções por Campylobacter/prevenção & controle , Campylobacter coli/fisiologia , Campylobacter jejuni/fisiologia , Feminino , Masculino , Doenças das Aves Domésticas/microbiologiaRESUMO
Bloodstream infections (BSIs) are considered a major cause of death worldwide. Staphylococcus spp. are one of the most BSIs prevalent bacteria, classified as high priority due to the increasing multidrug resistant strains. Thus, a fast, specific and sensitive method for detection of these pathogens is of extreme importance. In this study, we have designed a novel assay for detection of Staphylococcus in blood culture samples, which combines the advantages of a phage endolysin cell wall binding domain (CBD) as a specific probe with the accuracy and high-throughput of flow cytometry techniques. In order to select the biorecognition molecule, three different truncations of the C-terminus of Staphylococcus phage endolysin E-LM12, namely the amidase (AMI), SH3 and amidase+SH3 (AMI_SH3) were cloned fused with a green fluorescent protein. From these, a higher binding efficiency to Staphylococcus cells was observed for AMI_SH3, indicating that the amidase domain possibly contributes to a more efficient binding of the SH3 domain. The novel phage endolysin-based flow cytometry assay provided highly reliable and specific detection of 1-5 CFU of Staphylococcus in 10 mL of spiked blood, after 16 hours of enrichment culture. Overall, the method developed herein presents advantages over the standard BSIs diagnostic methods, potentially contributing to an early and effective treatment of BSIs.
Assuntos
Bacteriemia/diagnóstico , Citometria de Fluxo/métodos , Infecções Estafilocócicas/diagnóstico , Staphylococcus/isolamento & purificação , Bacteriemia/microbiologia , Proteínas de Bactérias/metabolismo , Limite de Detecção , Sondas Moleculares/metabolismo , Domínios Proteicos , Reprodutibilidade dos Testes , Infecções Estafilocócicas/microbiologia , Staphylococcus/metabolismo , Fagos de Staphylococcus/metabolismo , Proteínas Virais/metabolismoRESUMO
Studies involving antimicrobial-coated endotracheal tubes are scarce, and new approaches to control multidrug-resistant Pseudomonas aeruginosa biofilm on these devices should be investigated. In this study, five new P. aeruginosa bacteriophages from domestic sewage were isolated. All of them belong to the order Caudovirales, Myoviridae family. They are pH and heat stable and produce 27 to 46 particles after a latent period of 30 min at 37°C. Their dsDNA genome (ranging from â¼62 to â¼65 kb) encodes 65 to 89 different putative proteins. They exhibit a broad lytic spectrum and infect 69.7% of the P. aeruginosa strains tested. All the bacteriophages were able to reduce the growth of P. aeruginosa strains in planktonic form. The bacteriophages were also able to reduce the biofilm viability rates and the metabolic activity of P. aeruginosa strains in a model of biofilms associated with endotracheal tubes. In addition, scanning electron microscopy micrographs showed disrupted biofilms and cell debris after treatment of bacteriophages, revealing remarkable biofilm reduction. The lytic activity on multidrug-resistant P. aeruginosa biofilm indicates that the isolated bacteriophages might be considered as good candidates for therapeutic studies and for the application of bacteriophage-encoded products.
RESUMO
BACKGROUND: The Double-Layer Agar (DLA) technique is extensively used in phage research to enumerate and identify phages and to isolate mutants and new phages. Many phages form large and well-defined plaques that are easily observed so that they can be enumerated when plated by the DLA technique. However, some give rise to small and turbid plaques that are very difficult to detect and count. To overcome these problems, some authors have suggested the use of dyes to improve the contrast between the plaques and the turbid host lawns. It has been reported that some antibiotics stimulate bacteria to produce phages, resulting in an increase in final titer. Thus, antibiotics might contribute to increasing plaque size in solid media. RESULTS: Antibiotics with different mechanisms of action were tested for their ability to enhance plaque morphology without suppressing phage development. Some antibiotics increased the phage plaque surface by up to 50-fold. CONCLUSION: This work presents a modification of the DLA technique that can be used routinely in the laboratory, leading to a more accurate enumeration of phages that would be difficult or even impossible otherwise.
Assuntos
Antibacterianos/farmacologia , Bacteriófagos/isolamento & purificação , Ensaio de Placa Viral/métodos , Ágar , Bactérias/efeitos dos fármacos , Bactérias/virologiaRESUMO
Phages have shown a high biotechnological potential with numerous applications. The advent of high-resolution microscopy techniques aligned with omic and molecular tools are revealing innovative phage features and enabling new processes that can be further exploited for biotechnological applications in a wide variety of fields. This special issue is a collection of original and review articles focusing on the most recent advances in phage-based biotechnology with applications for human benefit.