RESUMEN

Enterococci are robust Gram-positive bacteria that pose a significant threat in healthcare settings due to antibiotic resistance, with vancomycin-resistant enterococci (VRE) most prominent. To tackle this issue, bacteriophages (bacterial viruses) can be exploited as they specifically and efficiently target bacteria. Here, we successfully isolated and characterised a set of novel phages: SHEF10, SHEF11, SHEF13, SHEF14, and SHEF16 which target E. faecalis (SHEF10,11,13), or E. faecium (SHEF13, SHEF14 & SHEF16) strains including a range of clinical and VRE isolates. Genomic analysis shows that all phages are strictly lytic and diverse in terms of genome size and content, quickly and effectively lysing strains at different multiplicity of infections. Detailed analysis of the broad host-range SHEF13 phage revealed the crucial role of the enterococcal polysaccharide antigen (EPA) variable region in its infection of E. faecalis V583. In parallel, the discovery of a carbohydrate-targeting domain (CBM22) found conserved within the three phage genomes indicates a role in cell surface interactions that may be important in phage-bacterial interactons. These findings advance our comprehension of phage-host interactions and pave the way for targeted therapeutic strategies against antibiotic-resistant enterococcal infections.

Asunto(s)

Bacteriófagos , Enterococcus faecalis , Genoma Viral , Especificidad del Huésped , Bacteriófagos/genética , Bacteriófagos/fisiología , Bacteriófagos/clasificación , Bacteriófagos/aislamiento & purificación , Enterococcus faecalis/virología , Enterococcus faecalis/genética , Enterococcus faecium/virología , Enterococcus faecium/genética , Enterococcus/virología , Enterococcus/genética , Enterococos Resistentes a la Vancomicina/virología , Enterococos Resistentes a la Vancomicina/genética , Infecciones por Bacterias Grampositivas/microbiología , Humanos

RESUMEN

Increase of the enteric bacteriophages (phage), components of the enteric virome, has been associated with the development of inflammatory bowel diseases. However, little is known about how a given phage contributes to the regulation of intestinal inflammation. In this study, we isolated a new phage associated with Enterococcus gallinarum, named phiEG37k, the level of which was increased in C57BL/6 mice with colitis development. We found that, irrespective of the state of inflammation, over 95% of the E. gallinarum population in the mice contained phiEG37k prophage within their genome and the phiEG37k titers were proportional to that of E. gallinarum in the gut. To explore whether phiEG37k impacts intestinal homeostasis and/or inflammation, we generated mice colonized either with E. gallinarum with or without the prophage phiEG37k. We found that the mice colonized with the bacteria with phiEG37k produced more Mucin 2 (MUC2) that serves to protect the intestinal epithelium, as compared to those colonized with the phage-free bacteria. Consistently, the former mice were less sensitive to experimental colitis than the latter mice. These results suggest that the newly isolated phage has the potential to protect the host by strengthening mucosal integrity. Our study may have clinical implication in further understanding of how bacteriophages contribute to the gut homeostasis and pathogenesis.

Asunto(s)

Bacteriófagos/clasificación , Colitis/microbiología , Enterococcus/patogenicidad , Mucina 2/metabolismo , Animales , Bacteriófagos/genética , Bacteriófagos/aislamiento & purificación , Colitis/inmunología , Modelos Animales de Enfermedad , Enterococcus/virología , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , Filogenia , Secuenciación Completa del Genoma

RESUMEN

Once overlooked as passive bystanders of the human intestinal microbiota, new evidence is shedding light on the importance of enterococci and their bacteriophages (phages) in shaping human health. Natural predators of enterococci, phages represent a narrow spectrum, precision targeting modality for the eradication of problematic enterococci within the microbiota or infected tissue. The identification of enterococcal phage receptors, absorption factors, and transcriptional responses following phage infection reveals a complex predator-prey relationship that modulates enterococcal cell surface architecture, susceptibility to antibiotics, and adaptation to host associated environments. Considering the dry up of contemporary antibiotic discovery pipelines in the pharmaceutical industry and a continued emergence of multidrug-resistant enterococci, enterococcal phages may serve as bonafide therapeutics. We highlight current advances in enterococcal phage biology with emphasis on recent breakthroughs in potential therapeutic applications that place enterococcal phages at the forefront of next-generation biologics.

Asunto(s)

Bacteriófagos/fisiología , Enterococcus/virología , Infecciones por Bacterias Grampositivas/terapia , Animales , Antibacterianos/farmacología , Bacteriófagos/genética , Farmacorresistencia Bacteriana , Enterococcus/efectos de los fármacos , Enterococcus/genética , Enterococcus/fisiología , Infecciones por Bacterias Grampositivas/microbiología , Interacciones Huésped-Patógeno , Humanos , Terapia de Fagos

RESUMEN

A functional genetic switch from the lactococcal bacteriophage TP901-1, deciding which of two divergently transcribing promoters becomes most active and allows this bi-stable decision to be inherited in future generations requires a DNA region of less than 1 kb. The fragment encodes two repressors, CI and MOR, transcribed from the PR and PL promoters respectively. CI can repress the transcription of the mor gene at three operator sites (OR, OL, and OD), leading to the immune state. Repression of the cI gene, leading to the lytic (anti-immune) state, requires interaction between CI and MOR by an unknown mechanism, but involving a CI:MOR complex. A consensus for putative MOR binding sites (OM sites), and a common topology of three OM sites adjacent to the OR motif was here identified in diverse phage switches that encode CI and MOR homologs, in a search for DNA sequences similar to the TP901-1 switch. The OR site and all putative OM sites are important for establishment of the anti-immune repression of PR, and a putative DNA binding motif in MOR is needed for establishment of the anti-immune state. Direct evidence for binding between CI and MOR is here shown by pull-down experiments, chemical crosslinking, and size exclusion chromatography. The results are consistent with two possible models for establishment of the anti-immune repression of cI expression at the PR promoter.

Asunto(s)

Bacteriófagos/genética , Lactococcus lactis/virología , Regiones Promotoras Genéticas/genética , Elementos Reguladores de la Transcripción/genética , Proteínas Represoras/genética , Transactivadores/genética , Proteínas Reguladoras y Accesorias Virales/genética , Bacteriófagos/crecimiento & desarrollo , Sitios de Unión/genética , ADN Viral/genética , Proteínas de Unión al ADN/genética , Enterococcus/virología , Regulación Viral de la Expresión Génica/genética , Genoma Viral/genética , Lactococcus lactis/genética , Lisogenia/genética , Regiones Operadoras Genéticas/genética , Proteínas Represoras/metabolismo , Staphylococcus/virología , Streptococcus/virología , Transactivadores/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo

RESUMEN

CRISPR-Cas adaptive immune systems protect bacteria and archaea against their invading genetic parasites, including bacteriophages/viruses and plasmids. In response to this immunity, many phages have anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas targeting. To date, anti-CRISPR genes have primarily been discovered in phage or prophage genomes. Here, we uncovered acr loci on plasmids and other conjugative elements present in Firmicutes using the Listeria acrIIA1 gene as a marker. The four identified genes, found in Listeria, Enterococcus, Streptococcus and Staphylococcus genomes, can inhibit type II-A SpyCas9 or SauCas9, and are thus named acrIIA16-19. In Enterococcus faecalis, conjugation of a Cas9-targeted plasmid was enhanced by anti-CRISPRs derived from Enterococcus conjugative elements, highlighting a role for Acrs in the dissemination of plasmids. Reciprocal co-immunoprecipitation showed that each Acr protein interacts with Cas9, and Cas9-Acr complexes were unable to cleave DNA. Northern blotting suggests that these anti-CRISPRs manipulate single guide RNA length, loading or stability. Mirroring their activity in bacteria, AcrIIA16 and AcrIIA17 provide robust and highly potent broad-spectrum inhibition of distinct Cas9 proteins in human cells (for example, SpyCas9, SauCas9, SthCas9, NmeCas9 and CjeCas9). This work presents a focused analysis of non-phage Acr proteins, demonstrating a role in horizontal gene transfer bolstered by broad-spectrum CRISPR-Cas9 inhibition.

Asunto(s)

Proteína 9 Asociada a CRISPR/antagonistas & inhibidores , Sistemas CRISPR-Cas , Transferencia de Gen Horizontal , Plásmidos/metabolismo , ARN Guía de Kinetoplastida/antagonistas & inhibidores , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Conjugación Genética , ADN/antagonistas & inhibidores , ADN/genética , ADN/metabolismo , Enterococcus/genética , Enterococcus/virología , Células HEK293 , Humanos , Listeria/genética , Listeria/virología , Plásmidos/química , Unión Proteica , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Staphylococcus/genética , Staphylococcus/virología , Streptococcus/genética , Streptococcus/virología

RESUMEN

Bacteriophages (phages) have been proposed as alternative therapeutics for the treatment of multidrug-resistant bacterial infections. However, there are major gaps in our understanding of the molecular events in bacterial cells that control how bacteria respond to phage predation. Using the model organism Enterococcus faecalis, we used two distinct genomic approaches, namely, transposon library screening and RNA sequencing, to investigate the interaction of E. faecalis with a virulent phage. We discovered that a transcription factor encoding a LytR family response regulator controls the expression of enterococcal polysaccharide antigen (epa) genes that are involved in phage infection and bacterial fitness. In addition, we discovered that DNA mismatch repair mutants rapidly evolve phage adsorption deficiencies, underpinning a molecular basis for epa mutation during phage infection. Transcriptomic profiling of phage-infected E. faecalis revealed broad transcriptional changes influencing viral replication and progeny burst size. We also demonstrate that phage infection alters the expression of bacterial genes associated with intra- and interbacterial interactions, including genes involved in quorum sensing and polymicrobial competition. Together, our results suggest that phage predation has the potential to influence complex microbial behavior and may dictate how bacteria respond to external environmental stimuli. These responses could have collateral effects (positive or negative) on microbial communities, such as the host microbiota, during phage therapy.IMPORTANCE We lack fundamental understanding of how phage infection influences bacterial gene expression and, consequently, how bacterial responses to phage infection affect the assembly of polymicrobial communities. Using parallel genomic approaches, we have discovered novel transcriptional regulators and metabolic genes that influence phage infection. The integration of whole-genome transcriptomic profiling during phage infection has revealed the differential regulation of genes important for group behaviors and polymicrobial interactions. Our work suggests that therapeutic phages could more broadly influence bacterial community composition outside their intended host targets.

Asunto(s)

Bacteriófagos/fisiología , Enterococcus/fisiología , Enterococcus/virología , Genómica , Interacciones Microbianas , Alelos , Evolución Biológica , Reparación de la Incompatibilidad de ADN , Replicación del ADN , Elementos Transponibles de ADN , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genómica/métodos , Mutación

RESUMEN

The bacteriophages of E. faecalis strains AIM06 (DSM100702) and SR14 (DSM100701) have previously been validated as human-specific microbial source tracking (MST) markers in Thailand. In this study, their spatial and temporal distribution in a freshwater river was investigated for the first time (n = 48). The abundance of enterococci as a standard microbial water quality parameter was evaluated by both the qPCR detection assay with primers and a hydrolysis probe according to the US EPA Method 1611 and the US EPA Method 1600 membrane filtration culture method. AIM06 and SR14 phages were detected by a double layer agar assay and were present in 87.5% and 81.3% of all samples with a co-presence of 92.9% of phage-positive samples. After spiking the representative phages, the ranges of recovery efficiencies were 57.9-99.6% and 49.6-99.9% (n = 48) for AIM06 and SR14 phages, respectively. The absolute abundance of AIM06 and SR14 phages ranged from 0.25 to 221.94 and from 0.25 to 76.66 PFU/100 mL, respectively. Enterococci DNA copies and CFU were detected in all samples ranging from 3.24 to 6.32 log10 copies/100 mL and 100.00 to 1593 CFU/100 mL, respectively. Enterococci in the qPCR assay also showed a moderate correlation with the culture method. The AIM06 and SR14 phage results indicated continuing human faecal pollution along the river with no significant different levels among stations. Interestingly, the higher levels of enterococci in downstream stations for both the qPCR and culture methods along with the significant correlation with other faecal indicator organisms and non-human MST markers implied non-human faecal pollution. In conclusion, this study provides insightful information that could lead to effective water quality management and public health risk reduction from exposure to faecally-contaminated water.

Asunto(s)

Bacteriófagos/aislamiento & purificación , Enterococcus/aislamiento & purificación , Contaminantes del Agua/aislamiento & purificación , ADN Bacteriano/análisis , Enterococcus/genética , Enterococcus/virología , Monitoreo del Ambiente/métodos , Heces , Humanos , Ríos/microbiología , Tailandia , Clima Tropical , Microbiología del Agua , Calidad del Agua , Abastecimiento de Agua

RESUMEN

Chronic wounds affect thousands of people worldwide, causing pain and discomfort to patients and represent significant economical burdens to health care systems. The treatment of chronic wounds is very difficult and complex, particularly when wounds are colonized by bacterial biofilms which are highly tolerant to antibiotics. Enterococcus faecium and Enterococcus faecalis are within the most frequent bacteria present in chronic wounds. Bacteriophages (phages) have been proposed as an efficient and alternative against antibiotic-resistant infections, as those found in chronic wounds. We have isolated and characterized two novel enterococci phages, the siphovirus vB_EfaS-Zip (Zip) and the podovirus vB_EfaP-Max (Max) to be applied during wound treatment. Both phages demonstrated lytic behavior against E. faecalis and E. faecium. Genome analysis of both phages suggests the absence of genes associated with lysogeny. A phage cocktail containing both phages was tested against biofilms formed in wound simulated conditions at a multiplicity of infection of 1.0 and a 2.5 log CFU.mL-1 reduction in the bacterial load after at 3 h of treatment was observed. Phages were also tested in epithelial cells colonized by these bacterial species and a 3 log CFU.mL-1 reduction was observed using both phages. The high efficacy of these new isolated phages against multi-species biofilms, their stability at different temperatures and pH ranges, short latent periods and non-cytotoxicity to epithelial cells suggest their therapeutic use to control infectious biofilms present in chronic wounds.

Asunto(s)

Bacteriófagos/fisiología , Biopelículas , Enterococcus/virología , Terapia de Fagos , Infección de Heridas/microbiología , Infección de Heridas/terapia , Células 3T3 , Animales , Bacteriólisis , Bacteriófagos/aislamiento & purificación , Bacteriófagos/ultraestructura , Supervivencia Celular , Células Epiteliales/virología , Genes Virales , Especificidad del Huésped , Humanos , Concentración de Iones de Hidrógeno , Ratones , Temperatura

RESUMEN

Coastal water quality is deteriorating worldwide. Water quality monitoring is therefore essential for public health risk evaluation and the management of water bodies. This study investigated the feasibility of using bacteriophages of Enterococcus faecalis as sewage-specific faecal indicators, together with physicochemical (dissolved oxygen, pH, temperature and total suspended solids) and biological parameters, to assess coastal water quality using multivariate analysis incorporating non-detects. The principal component and cluster analyses demonstrated that coastal water quality was mostly influenced by biological parameters, including Escherichia coli and total coliforms, which were found in all 31 sampling sites, and enterococci, which was found in all but two sampling sites. The enterococcal bacteriophages AIM06 and SR14 were detected in 17 and 18 samples at concentrations up to 1,815 and 2,790 PFU/100 mL, respectively. Both bacteriophages co-presented in approximately 80% of phage-positive samples, and the concentrations at each site were not significantly different. Overall, either bacteriophage could be used to differentiate high- and low-level coastal water pollution, as grouped by cluster analysis. This study is the first to investigate the suitability of sewage-specific bacteriophages of E. faecalis for monitoring coastal water quality and emphasises the importance of a multivariate analysis with non-detects to facilitate coastal water quality monitoring and management.

Asunto(s)

Bacteriófagos/crecimiento & desarrollo , Monitoreo del Ambiente/métodos , Microbiología del Agua , Contaminación del Agua/análisis , Enterococcus/virología , Heces , Agua de Mar/química , Agua de Mar/microbiología , Agua de Mar/virología , Aguas del Alcantarillado/microbiología , Aguas del Alcantarillado/virología , Calidad del Agua/normas

RESUMEN

Enterococcus faecalis bacteria have been recently reported for their ability to host bacteriophages that are specifically from human sewage, suggesting their application to track human fecal contamination in water resources. However, little is known about the survivability of sewage-specific enterococcal bacteriophages in various water matrices under ambient and storage conditions. In this study, bacteriophages that were derived from the Thailand-isolated E. faecalis strains AIM06 and SR14 exhibited morphologies consistent with the Siphoviridae, Podoviridae, and Myoviridae families. Four representative bacteriophages were separately spiked into environmental water samples (n = 7) comprising freshwater and seawater with low- and high-pollution (LF, HF, LS, and HS, respectively) levels, defined according to Thailand Water Quality Standards. All bacteriophages decayed fastest in HS or HF samples at 30 °C, reaching a 5-log10 reduction in 2.2 to 9.8 days, and slowest in LS samples, requiring 8.8 to 23.5 days. The decay rates were 5 to 53 times lower at a storage temperature of 5 °C. HF samples could be stored for as little as 2.5 days to prevent the decay of 50% of the phages. Myoviridae phages decayed faster than Siphoviridae phages and Podoviridae phages in most water matrices at 30 °C. Moreover, the decay rates were 1.8 to 92 times slower in filtered samples, emphasizing a strong role for water constituents, i.e., suspended solids and natural microorganisms, in phage persistence. This study emphasized that differential enterococcal bacteriophage persistence should be considered when planning the monitoring and interpreting of fecal sources by microbial source tracking.

Asunto(s)

Bacteriófagos/fisiología , Agua Dulce/virología , Agua de Mar/virología , Aguas del Alcantarillado/virología , Microbiología del Agua , Enterococcus/virología , Heces/virología , Humanos

RESUMEN

AIM: The aim of this study was to determine the morphological diversity and environmental survival of human-specific phages infecting Enterococcus faecium host strain MW47, to support their use as microbial source tracking (MST) markers. METHODS AND RESULTS: Twenty phages capable of infecting strain MW47 were propagated and their morphologies were determined using transmission electron microscopy (TEM), which revealed that a heterogeneous group of phages was able to infect strain MW47. Three distinct morphologies from two different families (Myoviridae and Siphoviridae) were observed. In situ inactivation experiments were subsequently conducted to determine their environmental persistence. CONCLUSION: The findings revealed a statistically significant link between morphology and the rate of inactivation, with phages belonging to the Myoviridae family demonstrating more rapid inactivation in comparison to those belonging to the Siphoviridae family. SIGNIFICANCE AND IMPACT OF STUDY: The results suggest that while Enterococcus MW47 phages appear to be a potentially valuable MST tools, significant variations in the persistence of the different phages mean that the approach should be used with caution, as this may adversely affect the reliability of the approach, especially when comparing MW47 phage levels or the presence across different matrices (e.g. levels in sediments or shellfish). This highlights the importance of elucidating the ecological characteristics of newly proposed MST markers before they are used in full-scale MST investigations.

Asunto(s)

Bacteriófagos/aislamiento & purificación , Bacteriófagos/ultraestructura , Enterococcus/virología , Humanos , Myoviridae/aislamiento & purificación , Myoviridae/ultraestructura , Reproducibilidad de los Resultados , Siphoviridae/aislamiento & purificación , Siphoviridae/ultraestructura , Virulencia

RESUMEN

High-throughput sequencing (HTS) is an effective tool for bacteriophage genome and its termini analysis. HTS technology parallelizes the sequencing process, producing thousands to millions of reads concurrently. Terminal information of a bacteriophage genome is important and basic knowledge for understanding the biology of the bacteriophage. We have created a high-occurrence reads as termini theory and developed practical methods to determine the bacteriophage genome termini, which is based on the large data of HTS. With this method, the termini of the bacteriophage genome can be efficiently and reliably identified as a by-product of bacteriophage genome sequencing, by solely analyzing the sequence statistics of the raw sequencing data (reads), without any further lab experiments.

Asunto(s)

Bacteriófagos/genética , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Bacteriófagos/aislamiento & purificación , Secuencia de Bases , Biología Computacional , ADN/genética , ADN Viral/aislamiento & purificación , Electroforesis en Gel de Agar , Enterococcus/virología , Genómica , Filogenia , Reproducibilidad de los Resultados , Replicación Viral

Asunto(s)

Humanos , Animales , Masculino , Femenino , Virulencia , Quirópteros , Enterococcus/virología , Heces , Genes/efectos de los fármacos , Farmacorresistencia Microbiana/efectos de los fármacos

RESUMEN

Bacteriophages are an attractive alternative to faecal indicator bacteria (FIB), particularly as surrogates of enteric virus fate and transport, due to their closer morphological and biological properties. Based on a review of published data, we summarize densities of coliphages (F+ and somatic), Bacteroides spp. and enterococci bacteriophages (phages) in individual human waste, raw wastewater, ambient fresh and marine waters and removal through wastewater treatment processes utilizing traditional treatments. We also provide comparisons with FIB and enteric viruses whenever possible. Lastly, we examine fate and transport characteristics in the aquatic environment and provide an overview of the environmental factors affecting their survival. In summary, concentrations of bacteriophages in various sources were consistently lower than FIB, but more reflective of infectious enteric virus levels. Overall, our investigation indicates that bacteriophages may be adequate viral surrogates, especially in built systems, such as wastewater treatment plants. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriophage are alternative fecal indicators that may be better surrogates for viral pathogens than fecal indicator bacteria (FIB). This report offers a summary of the existing literature concerning the utility of bacteriophage as indicators of viral presence (fecal sources and surface waters) and persistence (in built infrastructure and aquatic environments). Our findings indicate that bacteriophage levels in all matrices examined are consistently lower than FIB, but similar to viral pathogens. Furthermore, in built infrastructure (e.g. wastewater treatment systems) bacteriophage closely mimic viral pathogen persistence suggesting they may be adequate sentinels of enteric virus removal.

Asunto(s)

Colifagos/aislamiento & purificación , Enterovirus/aislamiento & purificación , Monitoreo del Ambiente/métodos , Aguas Residuales/análisis , Purificación del Agua , Bacteroides/virología , Enterococcus/virología , Heces/microbiología , Heces/virología , Humanos , Aguas Residuales/microbiología , Aguas Residuales/virología , Microbiología del Agua

RESUMEN

BACKGROUND: Enterococcus faecalis and Enterococcus faecium are typical enterococcal bacterial pathogens. Antibiotic resistance means that the identification of novel E. faecalis and E. faecium phages against antibiotic-resistant Enterococcus have an important impact on public health. In this study, the E. faecalis phage IME-EF4, E. faecium phage IME-EFm1, and both their hosts were antibiotic resistant. To characterize the genome termini of these two phages, a termini analysis theory was developed to provide a wealth of terminal sequence information directly, using only high-throughput sequencing (HTS) read frequency statistics. RESULTS: The complete genome sequences of phages IME-EF4 and IME-EFm1 were determined, and our termini analysis theory was used to determine the genome termini of these two phages. Results showed 9 bp 3' protruding cohesive ends in both IME-EF4 and IME-EFm1 genomes by analyzing frequencies of HTS reads. For the positive strands of their genomes, the 9 nt 3' protruding cohesive ends are 5'-TCATCACCG-3' (IME-EF4) and 5'-GGGTCAGCG-3' (IME-EFm1). Further experiments confirmed these results. These experiments included mega-primer polymerase chain reaction sequencing, terminal run-off sequencing, and adaptor ligation followed by run-off sequencing. CONCLUSION: Using this termini analysis theory, the termini of two newly isolated antibiotic-resistant Enterococcus phages, IME-EF4 and IME-EFm1, were identified as the byproduct of HTS. Molecular biology experiments confirmed the identification. Because it does not require time-consuming wet lab termini analysis experiments, the termini analysis theory is a fast and easy means of identifying phage DNA genome termini using HTS read frequency statistics alone. It may aid understanding of phage DNA packaging.

Asunto(s)

Bacteriófagos/genética , Enterococcus/virología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Secuencias Repetidas Terminales , Farmacorresistencia Bacteriana , Enterococcus/aislamiento & purificación , Enterococcus faecalis/aislamiento & purificación , Enterococcus faecalis/virología , Enterococcus faecium/aislamiento & purificación , Enterococcus faecium/virología , Genoma Viral , Humanos , Modelos Genéticos , Modelos Estadísticos

RESUMEN

Bacteriophage endolysins are bacterial cell wall degrading enzymes whose potential to fight bacterial infections has been intensively studied. Endolysins from Gram-positive systems are typically described as monomeric and as having a modular structure consisting of one or two N-terminal catalytic domains (CDs) linked to a C-terminal region responsible for cell wall binding (CWB). We show here that expression of the endolysin gene lys170 of the enterococcal phage F170/08 results in two products, the expected full length endolysin (Lys170FL) and a C-terminal fragment corresponding to the CWB domain (CWB170). The latter is produced from an in-frame, alternative translation start site. Both polypeptides interact to form the fully active endolysin. Biochemical data strongly support a model where Lys170 is made of one monomer of Lys170FL associated with up to three CWB170 subunits, which are responsible for efficient endolysin binding to its substrate. Bioinformatics analysis indicates that similar secondary translation start signals may be used to produce and add independent CWB170-like subunits to different enzymatic specificities. The particular configuration of endolysin Lys170 uncovers a new mode of increasing the number of CWB motifs associated to CD modules, as an alternative to the tandem repetition typically found in monomeric cell wall hydrolases.

Asunto(s)

Bacteriófagos/genética , Pared Celular/metabolismo , Endopeptidasas/química , Endopeptidasas/genética , Secuencia de Aminoácidos , Bacteriófagos/enzimología , Sitios de Unión , Biología Computacional , Endopeptidasas/metabolismo , Enterococcus/virología , Escherichia coli/genética , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Homología de Secuencia de Aminoácido

RESUMEN

AIM: Study sensitivity of nosocomial microbes--causative agents of post-operative infectious complications in surgical profile patients to bacteriophages. MATERIALS AND METHODS: Microbe isolates from biosubstrates of 223 surgical patients served as material. Microbes were isolated from blood, urine, wounds, intravascular catheters, abdominal cavity drainage, discharge of pharynx, trachea (343 strains). Phagolysability of strains was determined by Ott method. RESULTS: A good lysability of Pseudomonas aeruginosa, Klebsiella, Escherichia coli by specific complex and monophages and lower--of staphylococci and enterococci was found in the presence of 83% methicillin-resistant staphylococci strains and 94% Gram-negative bacteria strains producing extended spectrum beta-lactamases. CONCLUSION: Continuation of use of various bacteriophage preparations against Gram-negative bacteria and implementation of measures to adopt phage preparations to staphylococci and enterococci are perspective considering multiple resistance of microbes to antibiotics.

Asunto(s)

Bacteriófagos/fisiología , Enterococcus/virología , Escherichia coli/virología , Klebsiella pneumoniae/virología , Staphylococcus aureus Resistente a Meticilina/virología , Pseudomonas aeruginosa/virología , Adolescente , Adulto , Anciano , Antibacterianos/uso terapéutico , Bacteriemia/tratamiento farmacológico , Bacteriemia/microbiología , Bacteriófagos/aislamiento & purificación , Bacteriófagos/patogenicidad , Bacteriuria/tratamiento farmacológico , Bacteriuria/microbiología , Catéteres de Permanencia/microbiología , Niño , Preescolar , Enterococcus/efectos de los fármacos , Enterococcus/aislamiento & purificación , Escherichia coli/efectos de los fármacos , Escherichia coli/aislamiento & purificación , Femenino , Humanos , Lactante , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/aislamiento & purificación , Lisogenia/fisiología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Persona de Mediana Edad , Infecciones Oportunistas/tratamiento farmacológico , Infecciones Oportunistas/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/aislamiento & purificación , Procedimientos Quirúrgicos Operativos , Resistencia betalactámica/efectos de los fármacos , beta-Lactamasas/metabolismo

RESUMEN

No microbial source tracking tool satisfies all the characteristics of an ideal indicator of human fecal pollution. For this reason, the potential of Enterococcus faecalis phages (enterophages) as markers of this type of contamination was tested by using eight Enterococcus type strains as the possible hosts. The prevalence of enterophages in animal feces and domestic sewage were determined, as were the inactivation rates in raw sewage at 4 °C and surface and tap waters at 22 °C. Enterophages were exclusively detected in raw sewage (up to 66.0 plaque forming units (PFU)/100 mL), suggesting a strictly human origin; and exhibited inactivation rates of approximately 0.002 to 0.05, 0.3 to 0.5 and 0.4 to 1.4 log day(-1) in raw sewage and surface and tap waters, respectively, similar to those of previous reports on human enteric viruses under similar conditions. Interestingly, phages infecting other Enterococcus type strains were detected in both animal feces and domestic sewage in concentrations of up to 335.8 PFU/g and 96.0 PFU/100 mL, and certain phage isolates infected several of the strains tested. This clearly indicates the possible promiscuous nature of some Enterococcus phages and thus opens up the opportunity to further characterize these as indices of specific fecal sources.

Asunto(s)

Colifagos/aislamiento & purificación , Enterococcus/virología , Heces/microbiología , Contaminación del Agua/análisis , Animales , Biomarcadores , Colifagos/fisiología , Halogenación , Humanos , Factores de Tiempo , Eliminación de Residuos Líquidos , Microbiología del Agua , Contaminantes del Agua , Purificación del Agua/métodos

RESUMEN

AIMS: The objective was to assess the impacts of repairing a failing onsite wastewater treatment system (OWTS, i.e., septic system) as related to coastal microbial water quality. METHODS AND RESULTS: Wastewater, groundwater and surface water were monitored for environmental parameters, faecal indicator bacteria (total coliforms, Escherichia coli, enterococci) and the viral tracer MS2 before and after repairing a failing OWTS. MS2 results using plaque enumeration and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) often agreed, but inhibition limited the qRT-PCR assay sensitivity. Prerepair, MS2 persisted in groundwater and was detected in the nearby creek; postrepair, it was not detected. In groundwater, total coliform concentrations were lower and E. coli was not detected, while enterococci concentrations were similar to prerepair levels. E. coli and enterococci surface water concentrations were elevated both before and after the repair. CONCLUSIONS: Repairing the failing OWTS improved groundwater microbial water quality, although persistence of bacteria in surface water suggests that the OWTS was not the singular faecal contributor to adjacent coastal waters. A suite of tracers is needed to fully assess OWTS performance in treating microbial contaminants and related impacts on receiving waters. Molecular methods like qRT-PCR have potential but require optimization. SIGNIFICANCE AND IMPACT OF STUDY: This is the first before and after study of a failing OWTS and provides guidance on selection of microbial tracers and methods.

Asunto(s)

Enterobacteriaceae/fisiología , Enterococcus/fisiología , Monitoreo del Ambiente , Microbiología del Agua , Calidad del Agua , Carga Bacteriana , Enterobacteriaceae/genética , Enterobacteriaceae/aislamiento & purificación , Enterobacteriaceae/virología , Enterococcus/genética , Enterococcus/aislamiento & purificación , Enterococcus/virología , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Escherichia coli/fisiología , Escherichia coli/virología , Agua Subterránea/microbiología , Levivirus/fisiología

RESUMEN

This paper describes the isolation of Enterococcus host strains, for potential use as simple bacteriophage (phage)-based microbial source tracking (MST) tools. Presumptive Enterococcus host strains were isolated from cattle feces, raw municipal wastewater, agricultural runoff, and waters impacted by farms or wastewater treatment works (WWTW) in southern England, United Kingdom (UK). All enterococcal host strains (n = 390) were first screened for their ability to detect phage in samples of raw municipal wastewater and fecal material from cattle, pigs, and sheep. Host strains that detected phage (n = 147) were ranked according to both their specificity to a particular fecal source and also the number of phages (expressed as plaque-forming units, PFU) that they detected per milliliter of sample. Host strains that demonstrated host specificity and which detected phages at levels greater than 100 PFU/mL (n = 29) were further tested using additional fecal samples of human and nonhuman origin. The specificity and sensitivity of the enterococcal host strains were found to vary, ranging from 44 to 100% and from 17 to 83%, respectively. Most notably, seven strains exhibited 100% specificity to either cattle, human, or pig samples. Isolates exhibiting specificity to cattle were identified as belonging to the species Enterococcus casseliflavus , Enterococcus mundtii , or Enterococcus gallinarum , while human and pig isolates were members of either Enterococcus faecium or Enterococcus faecalis . The high specificity of phages infecting Enterococcus hosts and the simplicity and relatively low cost of the approach collectively indicate a strong potential for using this method as a tool in MST.

Asunto(s)

Bacteriófagos/patogenicidad , Enterococcus/virología , Monitoreo del Ambiente/métodos , Agua Dulce/microbiología , Contaminación del Agua/análisis , Crianza de Animales Domésticos , Enterococcus/clasificación , Enterococcus/aislamiento & purificación , Agua Dulce/virología , Reino Unido