Listeria Phage A511 Research Articles

Poly (lactic acid) and whey protein/pullulan composite bilayer film containing phage A511 as an anti-Listerial packaging for chicken breast at refrigerated temperatures

Listeria phage A511 were stabilized in whey protein concentrate/pullulan (WP) composite and poly (lactic acid) (PLA) bilayer films with different thickness ratios (100%, 70/30%, 50/50%, 30/70%). As the thickness of WP layer decreased, so did the phage recovery from the film. The highest phage recovery belonged to 30PLA/70WP, which was statistically similar to 100WP. Investigation of storage of phage-containing 100WP and 30PLA/70WP films for 60 days at 25 °C and 53% relative humidity revealed that films could maintain phage titers for up to 20 days. Phage-containing bilayer film was selected for studying chicken breast due to its better barrier, mechanical, and visual properties than 100WP and stability of these properties during storage. The maximum release of phages from 30PLA/70WP to the chicken breast fillet at 4 °C and 10 °C occurred after 24 h. The phage-containing films had inhibition effects on Listeria in chicken fillets similar to free phage treatment.

Development of whey protein concentrate/pullulan composite films containing bacteriophage A511: Functional properties and anti-Listerial effects during storage

The present research examined the stabilization of Listeria phage A511 in whey protein concentrate (WPC), pullulan (PULL) films and their composites. The functional properties of the films were investigated during 60 days of storage at 25 °C and relative humidity of 53%. Pure films of WPC and PULL were not successful in phage stabilization. The highest phage recovery at zero time belonged to 30WPC:70PULL and 50WPC:50PULL films. Phage distribution was uniform in composite films. Addition of phage to the films resulted in enhanced opacity, b and ∆E. Antimicrobial effects in the solid medium in 50WPC:50PULL and 70WPC:30PULL films were reduced to zero, and the opacity, b and ∆E of phage-containing films increased over 60 days. In 30WPC:70PULL film the elastic modulus and tensile strength were enhanced while the elongation at break was reduced over time. Overall, 30WPC: 70PULL film had the best performance of the physical, mechanical and anti-Listerial properties during storage.

Uncovering a hidden diversity: optimized protocols for the extraction of dsDNA bacteriophages from soil

BackgroundBacteriophages (phages) are the most numerous biological entities on Earth and play a crucial role in shaping microbial communities. Investigating the bacteriophage community from soil will shed light not only on the yet largely unknown phage diversity, but may also result in novel insights towards their functioning in the global biogeochemical nutrient cycle and their significance in earthbound ecosystems. Unfortunately, information about soil viromes is rather scarce compared to aquatic environments, due to the heterogeneous soil matrix, which rises major technical difficulties in the extraction process. Resolving these technical challenges and establishing a standardized extraction protocol is, therefore, a fundamental prerequisite for replicable results and comparative virome studies.ResultsWe here report the optimization of protocols for the extraction of phage DNA from agricultural soil preceding metagenomic analysis such that the protocol can equally be harnessed for phage isolation. As an optimization strategy, soil samples were spiked with Listeria phage A511 (Myovirus), Staphylococcus phage 2638AΔLCR (Siphovirus) and Escherichia phage T7 (Podovirus) (each 106 PFU/g soil). The efficacy of phage (i) elution, (ii) filtration, (iii) concentration and (iv) DNA extraction methods was tested. Successful extraction routes were selected based on spiked phage recovery and low bacterial 16S rRNA gene contaminants. Natural agricultural soil viromes were then extracted with the optimized methods and shotgun sequenced. Our approach yielded sufficient amounts of inhibitor-free viral DNA for shotgun sequencing devoid of amplification prior library preparation, and low 16S rRNA gene contamination levels (≤ 0.2‰). Compared to previously published protocols, the number of bacterial read contamination was decreased by 65%. In addition, 379 novel putative complete soil phage genomes (≤ 235 kb) were obtained from over 13,000 manually identified viral contigs, promising the discovery of a large, previously inaccessible viral diversity.ConclusionWe have shown a considerably enhanced extraction of the soil phage community by protocol optimization that has proven robust in both culture-dependent as well as through viromic analyses. Our huge data set of manually curated soil viral contigs substantially increases the amount of currently available soil virome data, and provides insights into the yet largely undescribed soil viral sequence space.

A functional type II-A CRISPR-Cas system from Listeria enables efficient genome editing of large non-integrating bacteriophage.

CRISPR–Cas systems provide bacteria with adaptive immunity against invading DNA elements including bacteriophages and plasmids. While CRISPR technology has revolutionized eukaryotic genome engineering, its application to prokaryotes and their viruses remains less well established. Here we report the first functional CRISPR–Cas system from the genus Listeria and demonstrate its native role in phage defense. LivCRISPR-1 is a type II-A system from the genome of L. ivanovii subspecies londoniensis that uses a small, 1078 amino acid Cas9 variant and a unique NNACAC protospacer adjacent motif. We transferred LivCRISPR-1 cas9 and trans-activating crRNA into Listeria monocytogenes. Along with crRNA encoding plasmids, this programmable interference system enables efficient cleavage of bacterial DNA and incoming phage genomes. We used LivCRISPR-1 to develop an effective engineering platform for large, non-integrating Listeria phages based on allelic replacement and CRISPR-Cas-mediated counterselection. The broad host-range Listeria phage A511 was engineered to encode and express lysostaphin, a cell wall hydrolase that specifically targets Staphylococcus peptidoglycan. In bacterial co-culture, the armed phages not only killed Listeria hosts but also lysed Staphylococcus cells by enzymatic collateral damage. Simultaneous killing of unrelated bacteria by a single phage demonstrates the potential of CRISPR–Cas-assisted phage engineering, beyond single pathogen control.

Encapsulation of Listeria Phage A511 by Alginate to Improve Its Thermal Stability.

Microencapsulation is a versatile method for enhancing the stability of bacteriophages under harsh conditions, such as those which occur during thermal processing. For food applications, encapsulation in food-grade polymer matrices is desirable owing to their nontoxicity and low cost. Here, we describe the encapsulation of Listeria phage A511 using sodium alginate, gum arabic, and gelatin to maximize its viability during thermal processing.

Characterization of antimicrobial properties of Salmonella phage Felix O1 and Listeria phage A511 embedded in xanthan coatings on Poly(lactic acid) films

Beyond simply providing a barrier between food and external contaminants, active packaging technologies aim to inhibit pathogen survival and growth within the packaged environment. Bacteriophages have a proven track record as targeted antimicrobials but have yet to be successfully integrated in active packaging without serious loss of activity. We have developed two bacteriophage based xanthan coatings on poly(lactic acid) (PLA) film which significantly inhibits Salmonella Typhimurium and Listeria monocytogenes growth in culture (P < 0.01), and significantly reduces survival and growth of diverse cocktails of Salmonella sp. and L. monocytogenes respectively on precooked sliced turkey breast over 30 days of anaerobic packaging at 4 or 10 °C (P < 0.05). Specifically reductions of 0.832 log at 4 °C and 1.30 log at 10 °C for Salmonella sp., and 6.31 log at 4 °C and 1.52 log at 10 °C for L. monocytogenes were observed. The coating containing Listeria phage A511 also significantly inhibited growth of L. monocytogenes over 14 days in aerobic packaging (3.79 log at 4 °C, 2.17 log at 10 °C, P < 0.05). These coatings showed 99.99% phage release within 30 min for both phages. Similar approaches could be used to develop packaging inhibitory to other significant foodborne pathogens such as Campylobacter, and Escherichia coli, as well as spoilage bacteria.

Listeria phage A511, a model for the contractile tail machineries of SPO1‐related bacteriophages

Recognition of the bacterial host and attachment to its surface are two critical steps in phage infection. Here we report the identification of Gp108 as the host receptor-binding protein of the broad host-range, virulent Listeria phage A511. The ligands for Gp108 were found to be N-acetylglucosamine and rhamnose substituents of the wall teichoic acids of the bacterial cell wall. Transmission electron microscopy and immunogold-labelling allowed us to create a model of the A511 baseplate in which Gp108 forms emanating short tail fibres. Data obtained for related phages, such as Staphylococcus phages ISP and Twort, demonstrate the evolutionary conservation of baseplate components and receptor-binding proteins within the Spounavirinae subfamily, and contractile tail machineries in general. Our data reveal key elements in the infection process of large phages infecting Gram-positive bacteria and generate insights into the complex adsorption process of phage A511 to its bacterial host.

Genome sequence and analysis of a broad-host range lytic bacteriophage that infects the Bacillus cereus group

BackgroundComparatively little information is available on members of the Myoviridae infecting low G+C content, Gram-positive host bacteria of the family Firmicutes. While numerous Bacillus phages have been isolated up till now only very few Bacillus cereus phages have been characterized in detail.ResultsHere we present data on the large, virulent, broad-host-range B. cereus phage vB_BceM_Bc431v3 (Bc431v3). Bc431v3 features a 158,618 bp dsDNA genome, encompassing 239 putative open reading frames (ORFs) and, 20 tRNA genes encoding 17 different amino acids. Since pulsed-field gel electrophoresis indicated that the genome of this phage has a mass of 155-158 kb Bc431v3 DNA appears not to contain long terminal repeats that are found in the genome of Bacillus phage SPO1.ConclusionsBc431v3 displays significant sequence similarity, at the protein level, to B. cereus phage BCP78, Listeria phage A511 and Enterococcus phage ØEF24C and other morphologically related phages infecting Firmicutes such as Staphylococcus phage K and Lactobacillus phage LP65. Based on these data we suggest that Bc431v3 should be included as a member of the Spounavirinae; however, because of all the diverse taxonomical information has been addressed recently, it is difficult to determine the genus. The Bc431v3 phage contains some highly unusual genes such as gp143 encoding putative tRNAHis guanylyltransferase. In addition, it carries some genes that appear to be related to the host sporulation regulators. These are: gp098, which encodes a putative segregation protein related to FstK/SpoIIIE DNA transporters; gp105, a putative segregation protein; gp108, RNA polymerase sigma factor F/B; and, gp109 encoding RNA polymerase sigma factor G.

Reporter bacteriophage A511::celBtransduces a hyperthermostable glycosidase fromPyrococcus furiosusfor rapid and simple detection of viable Listeria cells

Reporter bacteriophages for detection of pathogenic bacteria offer fast and sensitive screening for live bacterial targets. We present a novel strategy employing a gene encoding a hyperthermophilic enzyme, permitting the use of various substrates and assay formats. The celB gene from the hyperthermophilic archaeon Pyrococcus furiosus specifying an extremely thermostable β-glycosidase was inserted into the genome of the broad host range, virulent Listeria phage A511 by homologous recombination. It is expressed at the end of the infectious cycle, under control of the strong major capsid gene promoter Pcps. Infection of Listeria with A511::celB results in strong gene expression and synthesis of a fully functional β-glycosidase. The reporter phage was tested for detection of viable Listeria cells with different chromogenic, fluorescent or chemiluminescent substrates. The best signal-to-noise ratio and sufficiently high sensitivity was obtained using the inexpensive substrate 4-Methylumbelliferyl-α-D-Glucopyranoside (MUG). The reporter phage assay is simple to perform and can be completed in about 6 h. Phage infection, as well as the subsequent temperature shift, enzymatic substrate conversion and signal recordings are independent from each other and may be performed separately. The detection limit for viable Listeria monocytogenes in an assay format adapted to 96-well microplates was 7.2 × 10(2) cells per well, corresponding to 6 × 10(3) cfu per ml in suspension. Application of the A511::celB protocol to Listeria in spiked chocolate milk and salmon demonstrate the usefulness of the reporter phage for rapid detection of low numbers of the bacteria (10 cfu/g or less) in contaminated foods.

Bacteriophage biocontrol of Listeria monocytogenes on soft ripened white mold and red-smear cheeses.

Soft-ripened cheeses belong to the type of food most often contaminated with Listeria monocytogenes, and they have been implicated in several outbreaks of listeriosis. Bacteriophages represent an attractive way to combat foodborne pathogens without affecting other properties of the food. We used the broad host range, virulent Listeria phage A511 for control of L. monocytogenes during the production and ripening phases of both types of soft-ripened cheeses, white mold (Camembert-type) cheese, as well as washed-rind cheese with a red-smear surface (Limburger-type). The surfaces of young, unripened cheese were inoculated with 10(1)-10(3) cfu/cm(2)L. monocytogenes strains Scott A (serovar 4b) or CNL 10(3)/2005 (serovar 1/2a). Phage was applied at defined time points thereafter, in single or repeated treatments, at 3 × 10(8) or 1 × 10(9) pfu/cm(2). With Scott A (10(3) cfu/cm(2)) and a single dose of A511 (3 × 10(8) pfu/cm(2)) on camembert-type cheese, viable counts dropped 2.5 logs at the end of the 21 day ripening period. Repeated phage application did not further inhibit the bacteria, whereas a single higher dose (1 × 10(9) pfu/cm(2)) was found to be more effective. On red-smear cheese ripened for 22 days, Listeria counts were down by more than 3 logs. Repeated application of A511 further delayed re-growth of Listeria, but did not affect bacterial counts after 22 days. With lower initial Listeria contamination (10(1)-10(2) cfu/cm(2)), viable counts dropped below the limit of detection, corresponding to more than 6 logs reduction compared to the control. Our data clearly demonstrate the potential of bacteriophage for biocontrol of L. monocytogenes in soft cheese.

Genome of a virulent bacteriophage Lb338-1 that lyses the probiotic Lactobacillus paracasei cheese strain

There is a lack of fundamental knowledge about the influence of bacteriophage on probiotic bacteria and other commensals in the gut. Here, we present the isolation and morphological and genetic characterization of a virulent narrow-host-range bacteriophage, φLb338-1. This phage was isolated from fresh sewage and was shown to infect the probiotic cheese strain Lactobacillus paracasei NFBC 338. Electron microscopy studies revealed that φLb338-1 is a member of the Myoviridae family, with an isometric head, a medium-sized contractile tail, and a complex base plate. Genome sequencing revealed a 142-kb genome with 199 open reading frames. Putative functions could be assigned to 22% of the open reading frames; these had significant homology to genes found in the broad-host-range SPO1-like group of phages which includes the Enterococcus faecalis phage φEF24C, Listeria phage A511, and Lactobacillus plantarum phage LP65. Interestingly, no significant genomic similarity was observed between the phage and the probiotic host strain. Future studies will determine if the presence of bacteriophage φLb338-1 or others in the human or animal gut plays an antagonistic role against the probiotic effect of beneficial bacteria.

Mining GOLD and new model organisms in biotechnology

Mining GOLD and new model organisms in biotechnology

Organization and transcriptional analysis of the Listeria phage A511 late gene region comprising the major capsid and tail sheath protein genes cps and tsh.

A511 is a broad-host-range, virulent myovirus for Listeria monocytogenes. The genes encoding major structural proteins of the capsid (cps) and tail sheath (tsh) were mapped to a 10.15-kb late gene fragment. We have determined the complete nucleotide sequence of this region and confirmed the identities of Cps (48.7 kDa) and Tsh (61.3 kDa) by N-terminal amino acid sequencing of both proteins. In addition, nine other open reading frames were identified. On the basis of amino acid sequence homologies to known phage-encoded proteins, some putative functions and locations could be assigned to some of the deduced gene products. We present evidence that the cps product is proteolytically cleaved between Lys-23 and Ser-24 to yield the 444-residue polypeptide found in the mature viral capsid. We also found that the N-terminal methionine is absent from the mature tail sheath protein. cps and tsh are late genes; mRNAs first appear 15 to 20 min after infection of L. monocytogenes. Northern (RNA) hybridizations of total late mRNA with specific oligonucleotide probes were used to determine the sizes of respective transcripts. Primer extension analyses enabled the positive identification of six late promoters, which were found to differ from those identified in the chromosome of Listeria spp. The bulk of transcripts from cps and tsh arise from two phage promoters with identical 13-nucleotide sequences (TGCTAGATTATAG [core region underlined]) in the -10 region which we speculate determines specific and timed expression of these genes. A 123-nucleotide leader sequence at the 5' end of the cps transcript was predicted to form a strong secondary structure (deltaG=-40.7 kcal [-170.3 kJ]/mol). Out results show that the strongly expressed A511 cps and tsh genes are included in two separate gene clusters and are independently regulated at the transcriptional level.