Characterization and Application of a Lytic Phage D10 against Multidrug-Resistant Salmonella

Salmonella is a widely distributed foodborne pathogen that is a serious threat to human health. The accelerated development of drug resistance and the increased demand for natural foods invoke new biocontrol agents to limit contamination by multidrug-resistant (MDR) Salmonella strains. In this study, a lytic Salmonella phage named D10 was characterized at the biological and genomic levels. D10 possesses a short latent period (10 min) and a large burst size (163 PFU/cell), as well as adequate stability under a range of pH conditions and moderate thermal tolerance. D10 effectively lysed different MDR Salmonella serovars and repressed their dynamic growth in the medium. Genomic analysis disclosed that D10 is a new member of the Siphoviridae family and lacks the genes implicated in lysogeny, pathogenicity, or antibiotic resistance. A three-ingredient phage cocktail was then developed by mixing D10 with previously identified myovirus D1-2 and podovirus Pu20. The cocktail significantly reduced the count of MDR strains in liquid eggs, regardless of the temperature applied (4 and 25 °C). These results suggest that phage D10 is a promising tool to prevent food contamination by MDR Salmonella.

A Lytic Yersina pestis Bacteriophage Obtained From the Bone Marrow of Marmota himalayana in a Plague-Focus Area in China

A lytic Yersinia pestis phage vB_YpP-YepMm (also named YepMm for briefly) was first isolated from the bone marrow of a Marmota himalayana who died of natural causes on the Qinghai-Tibet plateau in China. Based on its morphologic (isometric hexagonal head and short non-contractile conical tail) and genomic features, we classified it as belonging to the Podoviridae family. At the MOI of 10, YepMm reached maximum titers; and the one-step growth curve showed that the incubation period of the phage was about 10 min, the rise phase was about 80 min, and the lysis amount of the phage during the lysis period of 80 min was about 187 PFU/cell. The genome of the bacteriophage YepMm had nucleotide-sequence similarity of 99.99% to that of the Y. pestis bacteriophage Yep-phi characterized previously. Analyses of the biological characters showed that YepMm has a short latent period, strong lysis, and a broader lysis spectrum. It could infect Y. pestis, highly pathogenic bioserotype 1B/O:8 Y. enterocolitica, as well as serotype O:1b Y. pseudotuberculosis—the ancestor of Y. pestis. It could be further developed as an important biocontrol agent in pathogenic Yersinia spp. infection.

TSP, a virulent Podovirus can control the growth of Staphylococcus aureus till 12 hours

Methicillin-resistant Staphylococcus aureus (MRSA) is a prevailing nosocomial pathogen that causes a large number of diseases in healthcare and community settings. The MRSA causes infections in different tissues of immunocompromised individuals leading to increased morbidity and mortality. It possess various virulence mechanisms to show resistance against to a lot of beta-lactam antibiotics. To tackle this emerging issue of MRSA, there is an urgent need of antibiotic alternatives and utilizing lytic bacteriophages is one of the best promising therapeutic approach. In the present study, a lytic bacteriophage TSP was isolated from hospital wastewater against MRSA. Its morphology, physiology, host specificity, burst size and lytic spectrum were determined and complete genome sequence was analyzed. TSP phage efficiently inhibit bacterial growth for up to 12 hours. TSP phage showed broad lytic spectrum against clinical isolates of MRSA (78%) and MSSA (37%). It showed stability at varying temperatures (25ºC, 37ºC) and pH (5–9), while its maximum storage stability was observed at 4ºC. It had short latent period (20min) and high burst size (103 PFU/ infected cell). TSP genome sequence and restriction analysis revealed that its genome is linear having 17,987 bp in length with an average GC content of 29.7%. The TSP genome showed 98% similarity to S aureus phages SCH1, SCH11 and vB SauP-436A1. According to comparative genomic analysis and phylogenetic tree analysis, TSP phage can be considered as a member of genus “P68viruses”. The strong lytic activity, broad host range and short latent period along with absence of any lysogenic and toxic genes make TSP a very good candidate for phage therapy against MRSA infections if prove safe during in vivo studies.

Isolation and Characterization of the First Freshwater Cyanophage Infecting Pseudanabaena

ABSTRACT Cyanobacteria are the major primary producers in both freshwater and marine environments. However, the majority of freshwater cyanophages remain unknown due to the limited number of cyanophage isolates. In this study, we present a novel lytic freshwater cyanophage, PA-SR01, which was isolated from the Singapore Serangoon Reservoir. To our knowledge, this is the first isolate of a cyanophage that has been found to infect the cyanobacterium Pseudanabaena. PA-SR01 has a narrow host range, a short latent period, and is chloroform sensitive. Distinct from the majority of cyanophage isolates, PA-SR01 has a tailless morphology. It is a double-stranded DNA virus with a 137,012-bp genome. Functional annotation for the predicted open reading frames (ORFs) of the PA-SR01 genome identified genes with putative functions related to DNA metabolism, structural proteins, lysis, host-derived metabolic genes, and DNA packaging. Out of 166 predicted ORFs, only 17 ORFs have homology with genes with known function. Phylogenetic analysis of the major capsid protein and terminase large subunit further suggests that phage PA-SR01 is evolutionary distinct from known cyanophages. Metagenomics sequence recruitment onto the PA-SR01 genome indicates that PA-SR01 represents a new evolutionary lineage of phage which shares considerable genetic similarities with phage sequences in aquatic environments and could play key ecological roles. IMPORTANCE This study presents the isolation of the very first freshwater cyanophage, PA-SR01, that infects Pseudanabaena, and fills an important knowledge gap on freshwater cyanophages as well as cyanophages infecting Pseudanabaena.

Characteristic of a Distant Relative of Teseptimavirus Genus Phages That Acquired the Ability to Lysogenize Its Host

Pseudomonas syringae is a plant pathogen, which groups over 40 pathovars. Climate change and international trade facilitate the worldwide spread of pathogenic P. syringae strains. In recent decades, infections with P. syringae have been causing large losses in vegetable growing and horticulture. With the aim to look for biocontrol agents that could minimize these losses, we isolated bacteriophages infective for certain P. syringae strains. One of these phages, designated by us as vB_PsyP_3MF5 (3MF5), appeared to have atypical properties. It formed clear plaques on the layers of sensitive cells at elevated temperatures but was unable to form plaques at room temperature. It quickly adsorbed to its host cells and had a short latent period and a large burst size at permissive temperature. However, several survivors of the phage infection could be isolated in a standard killing assay. They appeared to form a lysis zone when placed on a layer of cells that were not treated with this phage, indicating that they are 3MF5 lysogens. In support of that, their DNA could serve as a template for PCR amplification with 3MF5 specific primer pairs. The analysis of the 3MF5 genomic sequence (GenBank. Acc. No. MT597419) revealed features typical of Teseptimavirus genus phages which are obligatorily lytic and are unable to lysogenize cells. Additionally, comparative analysis of the predicted 3MF5 proteins excluded the presence of any obvious homolog of a typical phage repressor that inhibits transcription of early phage genes in lysogens. Conceivably, the repression is achieved either by the interaction of a temperature-sensitive host/phage protein with a region controlling the expression of phage early genes or by temperature-induced structural changes in phage RNA, which could act by the occlusion of ribosomal binding sites of early phage genes. Surprisingly, the results of our preliminary studies indicate that despite its conditionally temperate nature, 3MF5 exhibits biocontrol properties.

Identification of a newly isolated lytic bacteriophage against K24 capsular type, carbapenem resistant Klebsiella pneumoniae isolates

The increasing incidence of carbapenemase-producing K. pneumoniae strains (CP-Kps) in the last decade has become a serious global healthcare problem. Therapeutic options for the treatment of emerging hospital clones have drastically narrowed and therefore novel approaches must be considered. Here we have isolated and characterized a lytic bacteriophage, named vB_KpnS_Kp13, that was effective against all Verona integron-encoded metallo-β-lactamase (VIM) producing K. pneumoniae isolates originating from hospital samples (urine, blood, sputum and faeces), belonging to the ST15 clonal lineage and expressing the K24 capsule. Morphological characterization of vB_KpnS_Kp13 showed that the newly identified phage belonged to the Siphoviridae family, and phylogenetic analysis showed that it is part of a distinct clade of the Tunavirinae subfamily. Functional analysis revealed that vB_KpnS_Kp13 had relatively short latent period times (18 minutes) compared to other K. pneumoniae bacteriophages and could degrade biofilm by more than 50% and 70% in 24 and 48 hours respectively. Complete in vivo rescue potential of the new phage was revealed in an intraperitoneal mouse model where phages were administered intraperitoneally 10 minutes after bacterial challenge. Our findings could potentially be used to develop specific anti-CP-Kps bacteriophage-based therapeutic strategies against major clonal lineages and serotypes.

Characterization of the Three New Kayviruses and Their Lytic Activity Against Multidrug-Resistant Staphylococcus aureus

The development of antimicrobial resistance has become a global concern. One approach to overcome the problem of drug resistance is the application of bacteriophages. This study aimed at characterizing three phages isolated from sewage, which show lytic activity against clinical isolates of multidrug-resistant Staphylococcus aureus. Morphology, genetics and biological properties, including host range, adsorption rate, latent time, phage burst size and lysis profiles, were studied in all three phages. As analyzed by transmission electron microscopy (TEM), phages vB_SauM-A, vB_SauM-C, vB_SauM-D have a myovirion morphology. One of the tested phages, vB_SauM-A, has relatively rapid adsorption (86% in 17.5 min), short latent period (25 min) and extremely large burst size (~500 plaque-forming units (PFU) per infected cell). The genomic analysis revealed that vB_SauM-A, vB_SauM-C, vB_SauM-D possess large genomes (vB_SauM-A 139,031 bp, vB_SauM-C 140,086 bp, vB_SauM-D 139,088 bp) with low G+C content (~30.4%) and are very closely related to the phage K (95–97% similarity). The isolated bacteriophages demonstrate broad host range against MDR S. aureus strains, high lytic activity corresponding to strictly virulent life cycle, suggesting their potential to treat S. aureus infections.

An Unusually Short Latent Period of Therapy-Related Myeloid Neoplasm Harboring a Rare MLL-EP300 Rearrangement: Case Report and Literature Review

Therapy-related myeloid neoplasm (t-MN) is a late and lethal complication induced by chemotherapy and/or radiation therapy. Hematological malignancy is one of the most common primary diseases in patients with t-MN. However, the occurrence of t-MN in adult T-cell leukemia/lymphoma (ATL) patients is rarely reported, possibly due to the dismal prognosis of ATL per se. Here, we report a 62-year-old female who developed t-MN only three months after the completion of conventional chemotherapy and anti-CCR4 antibody for ATL acute type. The patient presented with persistent fever and monocytosis without any evidence of infectious diseases. Bone marrow examinations revealed chronic myelomonocytic leukemia-like disease with a chromosomal translocation of t(11;22)(q23;q13) as a solo cytogenetic abnormality, resulting in the diagnosis of t-MN. Next-generation sequencing analysis identified a rare chimeric transcript, MLL-EP300, without any additional somatic mutations. Although the patient underwent allogenic hematopoietic stem cell transplantation, she died of viral encephalomyelitis at 7 months after diagnosis of t-MN. Since recent therapeutic advances have extended the survival of patients with ATL, further evaluation of the long-term risks of developing t-MN in these patients is warranted.

Isolation and Characterization of ΦGF1, a Morphotype C3 Bacteriophage that InfectsEscherichia coli

ABSTRACTIt has been isolated a lytic bacteriophage specific toEscherichia coli, which can infect at least one different bacterial group. Phage ФGF1 was isolated from a wastewater treatment plant. It is resistant to the effect of chloroform and is stable at 40 and 50 °C. In addition, it is stable in the range of pH 5-8. Its host range is wide, infecting even strains from another genus such asShigella. The one-step growth curve yielded a short latent period of 15 minutes and a burst size of 85 PFU per infected cell. Under the electron microscope, this phage presents the C3 morphotype, extremely rare among members of the Podoviridae family. Phage ФGF1 shows some characteristics that could be considered useful in biocontrol applications againstE. coli. Keywords: Bacteriophage,Escherichia coli, morphotype C3,Podoviridae.IMPORTANCEWastewater throughout the world is a heavy carrier of potential pathogens that live in their environment along with other biological agents, such as bacteriophages, which play a controlling role of the bacterial populations there, as in soil. The description of the diversity of such bacteriophages is of paramount importance since they could be used to intentionally reduce or remove those pathogens from that environment. Our work describes a bacteriophage that lives primarily in this type of water.