Morphological, biological, and genomic characterization of a newly isolated lytic phage Sfk20 infecting Shigella flexneri, Shigella sonnei, and Shigella dysenteriae1.

Shigellosis, caused by Shigella bacterial spp., is one of the leading causes of diarrheal morbidity and mortality. An increasing prevalence of multidrug-resistant Shigella species has revived the importance of bacteriophages as an alternative therapy to antibiotics. In this study, a novel bacteriophage, Sfk20, has been isolated from water bodies of a diarrheal outbreak area in Kolkata (India) with lytic activity against many Shigella spp. Phage Sfk20 showed a latent period of 20 min and a large burst size of 123 pfu per infected cell in a one-step growth analysis. Phage-host interaction and lytic activity confirmed by phage attachment, intracellular phage development, and bacterial cell burst using ultrathin sectioning and TEM analysis. The genomic analysis revealed that the double-stranded DNA genome of Sfk20 contains 164,878 bp with 35.62% G + C content and 241 ORFs. Results suggested phage Sfk20 to include as a member of the T4 myoviridae bacteriophage group. Phage Sfk20 has shown anti-biofilm potential against Shigella species. The results of this study imply that Sfk20 has good possibilities to be used as a biocontrol agent.

Fluorometric detection of phages in liquid media: Application to turbid samples.

During the last years there has been a growing interest in the development of methods for phage detection and quantification in environmental, public health and industrial sectors. Good methods of phage monitoring contribute to progress in phage therapies, biocontrol and food safety studies. They have also been used to indicate the possible presence of microbiological hazards in drinking and recreational waters, and are an essential tool to prevent failure of microbe-based industrial bioreactors. Many of the sophisticated methods that have emerged to cover these needs are strongly hampered by the presence of turbidity in the samples, that results in decreased sensitivity. To avoid this, time consuming pretreatment steps must often be included that increase the overall complexity of the assays and the time required to perform them. With this in mind, we have explored an alternative method that fulfills the criteria of being simple, rapid and inexpensive and can be used to perform analysis in turbid media without any pretreatment steps. In this paper we develop a method that monitors lysis of an indicator culture when exposed to samples containing the target phage. The method is based on the properties of resazurin, a redox dye that becomes fluorescent when reduced by an active microbial culture. We analyzed the fluorescence kinetics of non-turbid phage-infected bacterial cultures as a function of both, phage abundance and initial cell concentration. For this purpose, different phage/host combinations were used and then, the addition of resazurin at different times (0, 30 and 60 min) was carefully evaluated for each phage/host combination, thus providing data for 168 combinations in total. Next, selected phage/host combinations were tested over 4 different turbidity models: 0, 1000, 2000 Nephelometric Turbidity Units (NTU) as well as in milk. The data obtained provided information about the duration of the assay and sensitivity thresholds in matrices with different turbidity grades. The results obtained indicate that the method can detect as few as 10 phage particles per assay volume within 3.5 h. If sensitivity is not an issue and the threshold of detection is increased to 107 phages the assay is considerably shortened, providing reliable results in only 40 min. Overall, the detection approach proposed in this work provides a simple, rapid and inexpensive solution that compares favorably, in terms of performance, with other high-end methods.

Isolation and Characterization T4- and T7-Like Phages that Infect the Bacterial Plant Pathogen Agrobacterium tumefaciens.

In the rhizosphere, bacteria-phage interactions are likely to have important impacts on the ecology of microbial communities and microbe-plant interactions. To better understand the dynamics of Agrobacteria-phage interactions, we have isolated diverse bacteriophages which infect the bacterial plant pathogen, Agrobacterium tumefaciens. Here, we complete the genomic characterization of Agrobacteriumtumefaciens phages Atu_ph04 and Atu_ph08. Atu_ph04-a T4-like phage belonging to the Myoviridae family-was isolated from waste water and has a 143,349 bp genome that encodes 223 predicted open reading frames (ORFs). Based on phylogenetic analysis and whole-genome alignments, Atu_ph04 is a member of a newly described T4 superfamily that contains other Rhizobiales-infecting phages. Atu_ph08, a member of the Podoviridae T7-like family, was isolated from waste water, has a 59,034 bp genome, and encodes 75 ORFs. Based on phylogenetic analysis and whole-genome alignments, Atu_ph08 may form a new T7 superfamily which includes Sinorhizobium phage PCB5 and Ochrobactrum phage POI1126. Atu_ph08 is predicted to have lysogenic activity, as we found evidence of an integrase and several transcriptional repressors with similarity to proteins in transducing phage P22. Together, this data suggests that Agrobacterium phages are diverse in morphology, genomic content, and lifestyle.

Novel T4 bacteriophages associated with black band disease in corals.

Research into causative agents underlying coral disease have focused primarily on bacteria, whereas potential roles of viruses have been largely unaddressed. Bacteriophages may contribute to diseases through the lysogenic introduction of virulence genes into bacteria, or prevent diseases through lysis of bacterial pathogens. To identify candidate phages that may influence the pathogenicity of black band disease (BBD), communities of bacteria (16S rRNA) and T4-bacteriophages (gp23) were simultaneously profiled with amplicon sequencing among BBD-lesions and healthy-coral-tissue of Montipora hispida, as well as seawater (study site: the central Great Barrier Reef). Bacterial community compositions were distinct among BBD-lesions, healthy coral tissue and seawater samples, as observed in previous studies. Surprisingly, however, viral beta diversities based on both operational taxonomic unit (OTU)-compositions and overall viral community compositions of assigned taxa did not differ statistically between the BBD-lesions and healthy coral tissue. Nonetheless, relative abundances of three bacteriophage OTUs, affiliated to Cyanophage PRSM6 and Prochlorococcus phages P-SSM2, were significantly higher in BBD-lesions than in healthy tissue. These OTUs associated with BBD samples suggest the presence of bacteriophages that infect members of the cyanobacteria-dominated BBD community, and thus have potential roles in BBD pathogenicity.

T4-like Escherichia coli phages from the environment carry blaCTX-M.

The resistance determinant blaCTX-M has many variants and has been the most commonly reported gene in clinical isolates of extended spectrum beta-lactamase producing Escherichia coli. Phages have been speculated as potential reservoirs of resistance genes and efficient vehicles for horizontal gene transfer. The objective of the study was to determine the prevalence and characterize bacteriophages that harbour the resistance determinant blaCTX-M . Escherichia coli specific bacteriophages were isolated from 15 samples including soil and water across Mangaluru, India using bacterial hosts that were sensitive to ß-lactams. Phenotypic and genotypic characterization based on plaque morphology, host range, restriction fragment length polymorphism (RFLP), presence of blaCTX-M and electron microscopy was performed. Of 36 phages isolated, seven were positive for Group 1 of blaCTX-M . Based on host range and RFLP pattern, the seven phages were classified into four distinct groups, each harbouring a variant of blaCTX-M . Five phages were T4-like Myoviridae by electron microscopy which was further confirmed by polymerase chain reaction (PCR) for T4 specific gp14. Generalized transduction of the CTX-M gene from these phages was also observed. The high prevalence (20%) of this gene blaCTX-M in the phage pool confirms the significant role of Myoviridae members, specifically T4-like phages in the dissemination of this resistance gene. SIGNIFICANCE AND IMPACT OF THE STUDY: The CTX-M gene that confers resistance to Beta-lactam class of drugs is widespread and diverse. Understanding mechanisms of antimicrobial resistance transfer is a key to devise methods for controlling it. Few studies indicate that bacteriophages are involved in the transfer of this gene but the type of phages involved and the degree of involvement remains to be explored. Our work has been able to identify the class of phages and the magnitude of involvement in the dissemination of this gene.

A T4virus prevents biofilm formation by Trueperella pyogenes.

Trueperella pyogenes is an opportunistic pathogen of many animal species. It causes economic losses worldwide, through mastitis, metritis and mainly endometritis in dairy cows. The ability of this bacterium to form biofilms is implicated in chronic infections through hampering immune system recognition and antibiotic penetration. Since it is difficult to eradicate T. pyogenes infections with antibiotics, phage therapy presents itself as a non-toxic, effective and economically viable alternative. The present study evaluated the use of the bacteriophage vB_EcoM-UFV13 (UFV13) in the prevention of T. pyogenes biofilm development. Based upon two different approaches (crystal violet and sessile cell counting) we observed that only a multiplicity of infection (MOI) of 10 showed a statistically significant reduction in biofilm formation. Although the exact mechanisms of biofilm disruption and cell-adhesion inhibition have not been determined, genome sequence analysis of the Escherichia phage UFV13 revealed a repertoire of virion-associated peptidoglycan hydrolases (VAPGHs). The present study presents new findings regarding the disruption of biofilm formation of a Gram-positive bacterium. Subsequent transcriptomic and proteomic research will help us to understand the exact interaction mechanisms between UFV13 and T. pyogenes.

Nuevos biomarcadores del asma grave / Novel Biomarkers in Severe Asthma

No disponible

The isolation and characterization of Stenotrophomonas maltophilia T4-like bacteriophage DLP6.

Increasing isolation of the extremely antibiotic resistant bacterium Stenotrophomonas maltophilia has caused alarm worldwide due to the limited treatment options available. A potential treatment option for fighting this bacterium is 'phage therapy', the clinical application of bacteriophages to selectively kill bacteria. Bacteriophage DLP6 (vB_SmoM-DLP6) was isolated from a soil sample using clinical isolate S. maltophilia strain D1571 as host. Host range analysis of phage DLP6 against 27 clinical S. maltophilia isolates shows successful infection and lysis in 13 of the 27 isolates tested. Transmission electron microscopy of DLP6 indicates that it is a member of the Myoviridae family. Complete genome sequencing and analysis of DLP6 reveals its richly recombined evolutionary history, featuring a core of both T4-like and cyanophage genes, which suggests that it is a member of the T4-superfamily. Unlike other T4-superfamily phages however, DLP6 features a transposase and ends with 229 bp direct terminal repeats. The isolation of this bacteriophage is an exciting discovery due to the divergent nature of DLP6 in relation to the T4-superfamily of phages.

[Isolation and phylogenetic analysis of major capsid gene (g23) of bacteriophages infecting Sinorhizobium meliloti].

Objective: In order to provide scientific data for studying the ecology of phage infecting Sinorhizobium meliloti, we examined morphological characteristics of rhizobiophages and their phylogenetic status of the major captain protein g23. Methods: Rhizobiophages were isolated by the double-layer plate method with host Sinorhizobium meliloti USDA1002T. The morphological characteristic of rhizobiophages were studied by transmission electron microscope. Meanwhile, rhizobiophage DNA was extracted, and the g23 that encodes the major capsid protein of bacteriophages was chosen as objective gene in PCR amplification. Results: Three rhizobiophages were isolated, all had an icosahedral head with approximately 81 to 86 nm in diameter and a long contractile tail with 54 to 70 nm in length. Basic local alignment search tool searches in website of national center for biotechnology information (NCBI) revealed that the g23 amino acid sequences obtained in this study had high identity with each other, but had very lower identity with those from T-evens, PseudoT-evens, SchizoT-evens and ExoT-evens. Phylogenetic analysis showed that the isolated g23 sequences formed a unique clade with those clones obtained from different ecosystem. Conclusion: All results indicated that the isolated rhizobiophages belong to family Myoviridae, a new group of T4 phages, which had lower identity with the g23 clones obtained in different environment.

Adsorption of T4 bacteriophages on planar indium tin oxide surface via controlled surface tailoring.

The work investigates the influence of surface physicochemical properties of planar indium tin oxide (ITO) as a model substrate on T4 bacteriophage adsorption. A comparative T4 bacteriophage adsorption study shows a significant difference in bacteriophage adsorption observed on chemically modified planar ITO when compared to similarly modified particulate ITO, which infers that trends observed in virus-particle interaction studies are not necessarily transferrable to predict virus-planar surface adsorption behaviour. We also found that ITO surfaces modified with methyl groups, (resulting in increased surface roughness and hydrophobicity) remained capable of adsorbing T4 bacteriophage. The adsorption of T4 onto bare, amine and carboxylic functionalised planar ITO suggests the presence of a unique binding behaviour involving specific functional groups on planar ITO surface beyond the non-specific electrostatic interactions that dominate phage to particle interactions. The paper demonstrates the significance of physicochemical properties of surfaces on bacteriophage-surface interactions.

The kinetics of Escherichia coli B growth and bacteriophage T4 multiplication in SM-1 novel minimal culture medium.

The aim of this study was to develop a minimal medium for the cultivation of Escherichia coli B, which could be especially suitable for the industrial propagation of bacteriophage T4. The new defined, minimal SM-1 culture medium, contains free amino acids as the only nitrogen source and enables the bacteria generation time to be prolonged and satisfactory phage titers to be achieved. The presence of organic ingredients, such as meat extracts, yeast hydrolysates, enzymatic protein hydrolysates, in a culture medium may cause problems in the case of bacteria or phage cultures for therapeutic purposes. In the present study, we introduce a new medium, together with some procedures and applications for its usage. We also present new kinetics of E. coli B growth. Some traits such as the lack of high molecular proteins, a bacterial growth comparable to that in a rich medium, and the cost effectiveness of the medium, makes it highly competitive with currently used microbiological media. The surprisingly high titers of bacteriophage T4 obtained in our experiments suggest that SM-1 medium has the potential to find a broad application in medicine, especially in infectious disease therapy, pharmacy and biotechnology.

Phage amplification and immunomagnetic separation combined with targeted mass spectrometry for sensitive detection of viable bacteria in complex food matrices.

We have developed and describe here for the first time a highly sensitive method for the fast and unambiguous detection of viable Escherichia coli in food matrices. The new approach is based on using label-free phages (T4), obligate parasites of bacteria, which are attractive for pathogen detection because of their inherent natural specificity and ease of use. A specific immunomagnetic separation was used to capture the progeny phages produced. Subsequently, T4 phage markers were detected by liquid chromatography coupled to targeted mass spectrometry. Combining the specificity of these three methodologies is of great interest in developing an alternative to conventional time-consuming culture-based technologies for the detection of viable bacteria for industrial applications. First, optimization experiments with phage T4 spiked in complex matrices (without a phage amplification event) were performed and demonstrated specific, sensitive, and reproducible phage capture and detection in complex matrices including Luria-Bertani broth, orange juice, and skimmed milk. The method developed was then applied to the detection of E. coli spiked in foodstuffs (with a phage amplification event). After having evaluated the impact of infection duration on assay sensitivity, we showed that our assay specifically detects viable E. coli in milk at an initial count of ≥1 colony-forming unit (cfu)/mL after an 8-h infection. This excellent detection limit makes our new approach an alternative to PCR-based assays for rapid bacterial detection.

Highly sensitive and specific detection of E. coli by a SERS nanobiosensor chip utilizing metallic nanosculptured thin films.

A nanobiosensor chip, utilizing surface enhanced Raman spectroscopy (SERS) on nanosculptured thin films (nSTFs) of silver, was shown to detect Escherichia coli (E. coli) bacteria down to the concentration level of a single bacterium. The sensor utilizes highly enhanced plasmonic nSTFs of silver on a silicon platform for the enhancement of Raman bands as checked with adsorbed 4-aminothiophenol molecules. T-4 bacteriophages were immobilized on the aforementioned surface of the chip for the specific capture of target E. coli bacteria. To demonstrate that no significant non-specific immobilization of other bacteria occurs, three different, additional bacterial strains, Chromobacterium violaceum, Paracoccus denitrificans and Pseudomonas aeruginosa were used. Furthermore, experiments performed on an additional strain of E. coli to address the specificity and reusability of the sensor showed that the sensor operates for different strains of E. coli and is reusable. Time resolved phase contrast microscopy of the E. coli-T4 bacteriophage chip was performed to study its interaction with bacteria over time. Results showed that the present sensor performs a fast, accurate and stable detection of E. coli with ultra-small concentrations of bacteria down to the level of a single bacterium in 10 µl volume of the sample.

An unconventional approach to impedance microbiology: detection of culture media conductivity variations due to bacteriophage generated lyses of host bacteria.

A novel and unconventional approach to impedance microbiology has been under investigation. In our approach, solution conductivity variations are generated from bacteriophage lyses of infected host cells and the consequent release of conductive endoplasmic material. To sensitively detect the lysis, low conductive growth media have been developed. A microchip has been fabricated to perform the analysis. The microchip is made of two bare gold electrodes and PDMS microchamber of 36 nL volume. Escherichia coli and selective phages T4 have been used as case study. Proof-of-principle experiments are here presented and discussed. The method was characterised in a wide range between 10(4) and 10(8) CFU/mL, where linear relation was found between conductivity variation and cell concentration in a log10 vs. log10 plot. The method is suited to integration with sample preparation based on phage-functionalised magnetic beads. It has a potential detection limit below 1 CFU/chamber and a total assay time of less than 1 h.

The genome, proteome and phylogenetic analysis of Sinorhizobium meliloti phage ΦM12, the founder of a new group of T4-superfamily phages.

Phage ΦM12 is an important transducing phage of the nitrogen-fixing rhizobial bacterium Sinorhizobium meliloti. Here we report the genome, phylogenetic analysis, and proteome of ΦM12, the first report of the genome and proteome of a rhizobium-infecting T4-superfamily phage. The structural genes of ΦM12 are most similar to T4-superfamily phages of cyanobacteria. ΦM12 is the first reported T4-superfamily phage to lack genes encoding class I ribonucleotide reductase (RNR) and exonuclease dexA, and to possess a class II coenzyme B12-dependent RNR. ΦM12's novel collection of genes establishes it as the founder of a new group of T4-superfamily phages, fusing features of cyanophages and phages of enteric bacteria.

The structure of Sinorhizobium meliloti phage ΦM12, which has a novel T=19l triangulation number and is the founder of a new group of T4-superfamily phages.

ΦM12 is the first example of a T=19l geometry capsid, encapsulating the recently sequenced genome. Here, we present structures determined by cryo-EM of full and empty capsids. The structure reveals the pattern for assembly of 1140 HK97-like capsid proteins, pointing to interactions at the pseudo 3-fold symmetry axes that hold together the asymmetric unit. The particular smooth surface of the capsid, along with a lack of accessory coat proteins encoded by the genome, suggest that this interface is the primary mechanism for capsid assembly. Two-dimensional averages of the tail, including the neck and baseplate, reveal that ΦM12 has a relatively narrow neck that attaches the tail to the capsid, as well as a three-layer baseplate. When free from DNA, the icosahedral edges expand by about 5nm, while the vertices stay at the same position, forming a similarly smooth, but bowed, T=19l icosahedral capsid.

Amplification and purification of T4-like escherichia coli phages for phage therapy: from laboratory to pilot scale.

We investigated the amplification and purification of phage preparations with respect to titer, contamination level, stability, and technical affordability. Using various production systems (wave bags, stirred-tank reactors, and Erlenmeyer flasks), we obtained peak titers of 10(9) to 10(10) PFU/ml for T4-like coliphages. Phage lysates could be sterilized through 0.22-µm membrane filters without titer loss. Phages concentrated by differential centrifugation were not contaminated with cellular debris or bacterial proteins, as assessed by electron microscopy and mass spectrometry, respectively. Titer losses occurred by high-speed pelleting of phages but could be decreased by sedimentation through a sucrose cushion. Alternative phage concentration methods are prolonged medium-speed centrifugation, strong anion-exchange chromatography, and ultrafiltration, but the latter still allowed elevated lipopolysaccharide contamination. T4-like phages could not be pasteurized but maintained their infectivity titer in the cold chain. In the presence of 10 mM magnesium ions, phages showed no loss of titer over 1 month at 30°C.

Evidence that the heterogeneity of a T4 population is the result of heritable traits.

Many bacteriophage populations display heterogeneity in their adsorption characteristics; a portion of the phage population remains free in solution throughout adsorption experiments (residual fraction). This residual fraction generally constitutes a minority of phages that exhibit significantly slower adsorption kinetics than the main phage stock (main fraction). While this phenomenon is likely the result of evolutionary driving forces, the present study demonstrates that the residual fraction is not always the result of phenotypic variations within a single genotype, as is generally thought. Experiments with phage T4 showed that two subgroups with distinct adsorption traits that were passed on to their progeny could be isolated from the original phage stock. Sequencing of genes involved in adsorption revealed two point mutations in gene 37 of residual fraction isolates, which resulted in modifications to the long tail-fiber, the organelle of attachment and host cell recognition. Adsorption studies consistently showed that T4 phage stocks amplified from residual fraction isolates had significantly lower adsorption efficiencies than those amplified from main fractions. The conducted experiments provide convincing evidence that the observed heterogeneity in T4 adsorption behavior is the result of conserved mutations to the phage genome and is not exclusively the result of phenotypic variations within the population. While it is believed high mutation rates exist to hasten phage adaptation, this study shows that this bet hedging strategy can also, in the short term, inadvertently handicap the phage's adsorption capabilities to a given host under normal infection conditions, resulting in the residual fraction observed in adsorption experiments.

A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display.

Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In 'competitive phage display' bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins.

High diversity and potential origins of T4-type bacteriophages on the surface of Arctic glaciers.

Tailed bacteriophages are the most abundant viruses in the biosphere. Here we examined the T4-type bacteriophage community inhabiting the surface of two glaciers in Svalbard. We used a molecular approach to target g23, the major capsid protein gene, to demonstrate that in the extreme cryoconite hole habitats the T4-type phages are surprisingly diverse. Phylogenetic analysis revealed that cryoconite hole sediments harbour a mixed phage community spanning multiple T4-type phage subgroups. The majority (71 %) of phage sequences clustered into three novel phylogenetically distinct groups, whilst the remainder clustered with known marine and soil derived phage sequences. The meltwater in cryoconite holes also contained a further distinct phage community which was related to previously detected marine phage variants. The ability of phages to move between marine and glacial habitats was tested in a transplantation experiment. Phages from the nearby marine fjord were found to be capable of initiating infection of supraglacial bacteria, suggesting suitable hosts could be found by non-native phages. Together this evidence suggests that the surface of glaciers contain both novel and cosmopolitan phages, some of which may have arrived in the cryosphere from other biomes.