SGP-C: A Broad Host Range Temperate Bacteriophage; Against Salmonella gallinarum

Salmonella gallinarum is a poultry restricted-pathogen causing fowl-typhoid disease in adult birds with mortality rates up-to 80% and exhibit resistance against commonly used antibiotics. In this current study, a temperate broad host range bacteriophage SGP-C was isolated against S. gallinarum from poultry digesta. It showed infection ability in all the 15 tested field strains of S. gallinarum. The SGP-C phage produced circular, turbid plaques with alternate rings. Its optimum activity was observed at pH 7.0 and 37–42°C, with a latent period of 45 min and burst size of 187 virions/bacterial cell. The SGP-C lysogens, SGPC-L5 and SGPC-L6 exhibited super-infection immunity against the same phage, an already reported feature of lysogens. A virulence index of 0.5 and 0.001 as MV50 of SGP-C suggests its moderate virulence. The genome of SGP-C found circular double stranded DNA of 42 Kbp with 50.04% GC content, which encodes 63 ORFs. The presence of repressor gene at ORF49, and absence of tRNA sequence in SGP-C genome indicates its lysogenic nature. Furthermore, from NGS analysis of lysogens we propose that SGP-C genome might exist either as an episome, or both as integrated and temporary episome in the host cell and warrants further studies. Phylogenetic analysis revealed its similarity with Salmonella temperate phages belonging to family Siphoviridae. The encoded proteins by SGP-C genome have not showed homology with any known toxin and virulence factor. Although plenty of lytic bacteriophages against this pathogen are already reported, to our knowledge SGP-C is the first lysogenic phage against S. gallinarum reported so far.

An approach for slow distributed denial of service attack detection and alleviation in software defined networks

Over the last few years, the need for programmable networks has captured the interest of industrialists and academicians. It has led to the development of a paradigm called software defined network (SDN). It separates the network intelligence into the control plane and forwarding logic into the data plane. This architecture gives scope to various security issues of which denial of service (DoS) is the most common and challenging to detect. This paper focuses on the detection and mitigation of a slow DoS attack called Slowloris on Apache2 server in SDN based networks. The proposed solution is called Slowloris detection and mitigation mechanism (SDMM). Mininet, an emulator, and SimpleHTTPServer are used for simulation and the same is implemented using Zodiac FX OpenFlow switch, Ryu controller and Apache2 server. SDMM algorithm detects and mitigates prolonged Slowloris attack in typical networks as well as in slow networks with low bandwidth and high delay in 240-280s with an accuracy of 100% and 98% respectively. It uses expectation of burst size as a key factor for detection.

Phenotypic and Genomic Comparison of Klebsiella pneumoniae Lytic Phages: vB_KpnM-VAC66 and vB_KpnM-VAC13

Klebsiella pneumoniae is a human pathogen that worsens the prognosis of many immunocompromised patients. Here, we annotated and compared the genomes of two lytic phages that infect clinical strains of K. pneumoniae (vB_KpnM-VAC13 and vB_KpnM-VAC66) and phenotypically characterized vB_KpnM-VAC66 (time of adsorption of 12 min, burst size of 31.49 ± 0.61 PFU/infected cell, and a host range of 20.8% of the tested strains). Transmission electronic microscopy showed that vB_KpnM-VAC66 belongs to the Myoviridae family. The genomic analysis of the phage vB_KpnM-VAC66 revealed that its genome encoded 289 proteins. When compared to the genome of vB_KpnM-VAC13, they showed a nucleotide similarity of 97.56%, with a 93% of query cover, and the phylogenetic study performed with other Tevenvirinae phages showed a close common ancestor. However, there were 21 coding sequences which differed. Interestingly, the main differences were that vB_KpnM-VAC66 encoded 10 more homing endonucleases than vB_KpnM-VAC13, and that the nucleotidic and amino-acid sequences of the L-shaped tail fiber protein were highly dissimilar, leading to different three-dimensional protein predictions. Both phages differed significantly in their host range. These viruses may be useful in the development of alternative therapies to antibiotics or as a co-therapy increasing its antimicrobial potential, especially when addressing multidrug resistant (MDR) pathogens.

Kinetic characteristics of transcriptional bursting in a complex gene model with cyclic promoter structure

While transcription occurs often in a bursty manner, various possible regulations can lead to complex promoter patterns such as promoter cycles, giving rise to an important issue: How do promoter kinetics shape transcriptional bursting kinetics? Here we introduce and analyze a general model of the promoter cycle consisting of multi-OFF states and multi-ON states, focusing on the effects of multi-ON mechanisms on transcriptional bursting kinetics. The derived analytical results indicate that bust size follows a mixed geometric distribution rather than a single geometric distribution assumed in previous studies, and ON and OFF times obey their own mixed exponential distributions. In addition, we find that the multi-ON mechanism can lead to bimodal burst-size distribution, antagonistic timing of ON and OFF, and diverse burst frequencies, each further contributing to cell-to-cell variability in the mRNA expression level. These results not only reveal essential features of transcriptional bursting kinetics patterns shaped by multi-state mechanisms but also can be used to the inferences of transcriptional bursting kinetics and promoter structure based on experimental data.

Characteristics of a Bacteriophage, vB_Kox_ZX8, Isolated From Clinical Klebsiella oxytoca and Its Therapeutic Effect on Mice Bacteremia

Klebsiella oxytoca is an important nosocomial and community-acquired opportunistic pathogenic Klebsiella and has become the second most prevalent strain in the clinic after K. pneumoniae. However, there have been few reports of bacteriophages used for treating K. oxytoca. In this study, a novel bacteriophage, vB_Kox_ZX8, which specifically infects K. oxytoca AD3, was isolated for the first time from human fecal samples. The biological characteristics of vB_Kox_ZX8 showed an incubation period of 10 min, a burst size of 74 PFU/cell, and a stable pH range of 3–11. Genomic bioinformatics studies of vB_Kox_ZX8 showed that it belongs to the genus Przondovirus, subfamily Studiervirinae, family Autographiviridae. The genome of vB_Kox_ZX8 is 39,398 bp in length and contains 46 putative open reading frames encoding functional proteins, such as DNA degradation, packaging, structural, lysin-holin, and hypothetical proteins. We further investigated the efficacy of vB_Kox_ZX8 phage in the treatment of mice with bacteremia caused by K. oxytoca infection. The results showed that vB_Kox_ZX8 (5 × 109 PFU/mouse) injected intraperitoneally alone was metabolized rapidly in BALB/c mice, and no significant side effects were observed in the control and treatment groups. Importantly, intraperitoneal injection with a single dose of phage vB_Kox_ZX8 (5 × 107 PFU/mouse) for 1 h post-infection saved 100% of BALB/c mice from bacteremia induced by intraperitoneal challenge with a minimum lethal dose of K. oxytoca AD3. However, all negative control mice injected with PBS alone died. Owing to its good safety, narrow host infectivity, high lysis efficiency in vitro, and good in vivo therapeutic effect, phage vB_Kox_ZX8 has the potential to be an excellent antibacterial agent for clinical K. oxytoca-caused infections.

Characterization of N4-like Pseudomonas Phage vB_Pae-PA14 Isolated from Seawater Sampled in Thailand

Bacteriophage, a predator virus of bacteria, is an abundant biological entity in the biosphere. With ultimate applications in medicine and biotechnology, new phages are extensively being isolated and characterized. The objective of the present study was to characterize lytic bacteriophage vB_Pae-PA14 infecting Pseudomonas aeruginosa ATCC 27853 that was isolated from seawater in Thailand. vB_Pae-PA14 was subjected to whole genome phylogenetic analysis, host range test, biofilm test and characterization. Results showed that the phage belonged to a group of N4-like viruses, could infect P. aeruginosa isolates including carbapenem-resistant P. aeruginosa. The burst size of vB_Pae-PA14 was 86 plaque-forming unit/infected cells. Also, the phage showed a greater ability to control planktonic P. aeruginosa cells than the biofilm cells. Phage could withstand physical stresses especially the high salt concentration. In brief, lytic bacteriophage vB_Pae-PA14 infecting P. aeruginosa was isolated and characterized, which might be useful in further bacteriophage lytic applications.

Isolation, phenotypic characterization and comparative genomic analysis of 2019SD1, a polyvalent enterobacteria phage

Shigella has the remarkable capability to acquire antibiotic resistance rapidly thereby posing a significant public health challenge for the effective treatment of dysentery (Shigellosis). The phage therapy has been proven as an effective alternative strategy for controlling Shigella infections. In this study, we illustrate the isolation and detailed characterization of a polyvalent phage 2019SD1, which demonstrates lytic activity against Shigella dysenteriae, Escherichia coli, Vibrio cholerae, Enterococcus saccharolyticus and Enterococcus faecium. The newly isolated phage 2019SD1 shows adsorption time < 6 min, a latent period of 20 min and burst size of 151 PFU per bacterial cell. 2019SD1 exhibits considerable stability in a wide pH range and survives an hour at 50 °C. Under transmission electron microscope, 2019SD1 shows an icosahedral capsid (60 nm dia) and a 140 nm long tail. Further, detailed bioinformatic analyses of whole genome sequence data obtained through Oxford Nanopore platform revealed that 2019SD1 belongs to genus Hanrivervirus of subfamily Tempevirinae under the family Drexlerviridae. The concatenated protein phylogeny of 2019SD1 with the members of Drexlerviridae taking four genes (DNA Primase, ATP Dependent DNA Helicase, Large Terminase Protein, and Portal Protein) using the maximum parsimony method also suggested that 2019SD1 formed a distinct clade with the closest match of the taxa belonging to the genus Hanrivervirus. The genome analysis data indicate the occurrence of putative tail fiber proteins and DNA methylation mechanism. In addition, 2019SD1 has a well-established anti-host defence system as suggested through identification of putative anti-CRISPR and anti-restriction endonuclease systems thereby also indicating its biocontrol potential.

Impact of Phage CDHS-1 on the Transcription, Physiology and Pathogenicity of a Clostridioides difficile Ribotype 027 Strain, R20291

All known Clostridioides difficile phages encode integrases rendering them potentially able to lyse or lysogenise bacterial strains. Here, we observed the infection of the siphovirus, CDHS-1 on a ribotype 027 strain, R20291 and determined the phage and bacterial gene expression profiles, and impacts of phage infection on bacterial physiology and pathogenicity. Using RNA-seq and RT-qPCR we analysed transcriptomic changes during early, mid-log and late phases of phage replication at an MOI of 10. The phage has a 20 min latent period, takes 80 min to lyse cells and a burst size of ~37. All phage genes are highly expressed during at least one time point. The Cro/C1-transcriptional regulator, ssDNA binding protein and helicase are expressed early, the holin is expressed during the mid-log phase and structural proteins are expressed from mid-log to late phase. Most bacterial genes, particularly the metabolism and toxin production/regulatory genes, were downregulated from early phage replication. Phage-resistant strains and lysogens showed reduced virulence during Galleria mellonella colonization as ascertained by the larval survival and expression of growth (10), reproduction (2) and infection (2) marker genes. These data suggest that phage infection both reduces colonization and negatively impacts bacterial pathogenicity, providing encouraging data to support the development of this phage for therapy to treat C. difficile infection.

Isolation and Identification of a Siphoviridae Bacteriophage Targeting Multidrug-resistant Klebsiella pneumoniae

Background: Based on the WHO, multidrug-resistant Klebsiella pneumoniae is a priority pathogen that causes opportunistic infections and is widely spread in the environment. Phage therapy is considered a natural, safe, and very efficient alternative to treat difficult-to-treat infections. Objectives: This study aimed to isolate highly virulent, lytic bacteriophages and evaluate their efficacy for lysing multidrug-resistant K. pneumoniae. Methods: Municipal wastewater samples were collected and filtered using 0.22 µm syringe filters and cultivated with log-phase cultures of K. pneumoniae using enrichment media. After 48 h of incubation, the cultures were centrifuged, and the resultant supernatant was filtered (0.22 µm). The detection of the phage was done using the spot assay with K. pneumoniae as the host. One-step growth kinetics and bacterial reduction tests were conducted to assess the growth kinetics of the isolated phage. The stability of the isolated phage was characterized by subjecting it to various temperature and pH conditions. The chemical stability of the K. pneumoniae phage was determined by exposing it to various organic compounds. A panel of 20 bacterial strains was tested using the spot assay, as well as double agar overlying assay, to determine the host range of the isolated phage. Results: Out of 40 wastewater samples tested, only one sample was tested positive for the K. pneumoniae phage (2.5%) that was lytic against the host strain. The K. pneumoniae phage had a latent period of 15 min and a burst size of 100 virions per infected cell. It was most stable at 37°C and pH range of 6.0 to 10.0. Chemically, the K. pneumoniae phage was resistant to 10% chloroform treatment. Transmission electron micrograph indicated that the K. pneumoniae phage belonged to the order Caudovirales, family Siphoviridae, morphotype B1. Conclusions: Most of the characteristic features of the K. pneumoniae phage indicated the potential of this phage to be used in phage therapy. Hence, a comprehensive study is highly recommended to characterize the K. pneumoniae phage genome, detect its molecular interactions with the host cell, and determine its lytic activity in combination with other phages, which may lead to the efficient utilization of this phage in phage therapy against K. pneumoniae infections.

Efficacious antibacterial potency of novel bacteriophages against ESBL-producing Klebsiella pneumoniae isolated from burn wound infections

Background and Objectives: Prevalence of extended spectrum β-lactamase (ESBL) leads to the development of antibi- otic resistance and mortality in burn patients. One of the alternative strategies for controlling ESBL bacterial infections is clinical trials of bacteriophage therapy. The aim of this study was to isolate and characterize specific bacteriophages against ESBL-producing Klebsiella pneumoniae in patients with burn ulcers. Materials and Methods: Clinical samples were isolated from the hospitalized patient in burn medical centers, Iran. Bio- chemical screenings and 16S rRNA gene sequencing were determined. The phages were isolated from municipal sewerage treatment plants, Isfahan, Iran. TEM and FESEM, adsorption velocity, growth curve, host range, and the viability of the phage particles as well as proteomics and enzyme digestion patterns were examined. Results: The results showed that Klebsiella pneumoniae Iaufa_lad2 (GenBank accession number: MW836954) was con- firmed as an ESBL-producing strain using combined disk method. This bacterium showed significant sensitivity to three phages including PɸBw-Kp1, PɸBw-Kp2, and PɸBw-Kp3. Morphological characterization demonstrated that the phage PɸBw-Kp3 to the Siphoviridae family (lambda-like phages) and both phages PɸBw-Kp1 and ɸBw-Kp2 to the Podoviridae family (T1-like phages). The isolated bacteriophages had a large burst size, thermal and pH viability and efficient adsorption rate to the host cells. Conclusion: In present study, the efficacy of bacteriophages against ESBL pathogenic bacterium promises a remarkable achievement for phage therapy. It seems that, these isolated bacteriophages, in the form of phage cocktails, had a strong an- tibacterial impacts and a broad-spectrum strategy against ESBL-producing Klebsiella pneumoniae isolated from burn ulcers.