Bacteriophages of Thermophilic ‘Bacillus Group’ Bacteria—A Review

Bacteriophages of thermophiles are of increasing interest owing to their important roles in many biogeochemical, ecological processes and in biotechnology applications, including emerging bionanotechnology. However, due to lack of in-depth investigation, they are underrepresented in the known prokaryotic virosphere. Therefore, there is a considerable potential for the discovery of novel bacteriophage-host systems in various environments: marine and terrestrial hot springs, compost piles, soil, industrial hot waters, among others. This review aims at providing a reference compendium of thermophages characterized thus far, which infect the species of thermophilic ‘Bacillus group’ bacteria, mostly from Geobacillus sp. We have listed 56 thermophages, out of which the majority belong to the Siphoviridae family, others belong to the Myoviridae and Podoviridae families and, apparently, a few belong to the Sphaerolipoviridae, Tectiviridae or Corticoviridae families. All of their genomes are composed of dsDNA, either linear, circular or circularly permuted. Fourteen genomes have been sequenced; their sizes vary greatly from 35,055 bp to an exceptionally large genome of 160,590 bp. We have also included our unpublished data on TP-84, which infects Geobacillus stearothermophilus (G. stearothermophilus). Since the TP-84 genome sequence shows essentially no similarity to any previously characterized bacteriophage, we have defined TP-84 as a new species in the newly proposed genus Tp84virus within the Siphoviridae family. The information summary presented here may be helpful in comparative deciphering of the molecular basis of the thermophages’ biology, biotechnology and in analyzing the environmental aspects of the thermophages’ effect on the thermophile community.

A New Enterobacter cloacae Bacteriophage EC151 Encodes the Deazaguanine DNA Modification Pathway and Represents a New Genus within the Siphoviridae Family

A novel Enterobacter cloacae phage, EC151, was isolated and characterized. Electron microscopy revealed that EC151 has a siphovirus-like virion morphology. The EC151 nucleotide sequence shows limited similarity to other phage genomes deposited in the NCBI GenBank database. The size of the EC151 genome is 60,753 bp and contains 58 putative genes. Thirty-nine of them encode proteins of predicted function, 18 are defined as hypothetical proteins, and one ORF identifies as the tRNA-Ser-GCT-encoding gene. Six ORFs were predicted to be members of the deazaguanine DNA modification pathway, including the preQ0 transporter. Comparative proteomic phylogenetic analysis revealed that phage EC151 represents a distinct branch within a group of sequences containing clades formed by members of the Seuratvirus, Nonagvirus, and Vidquintavirus genera. In addition, the EC151 genome showed gene synteny typical of the Seuratvirus, Nonagvirus, and Nipunavirus phages. The average genetic distances of EC151/Seuratvirus, EC151/Nonagvirus, and EC151/Vidquintavirus are approximately equal to those between the Seuratvirus, Nonagvirus, and Vidquintavirus genera (~0.7 substitutions per site). Therefore, EC151 may represent a novel genus within the Siphoviridae family. The origin of the deazaguanine DNA modification pathway in the EC151 genome can be traced to Escherichia phages from the Seuratvirus genus.

A Novel Lytic Phage SZW_AS01 of Human Gut Bacteria Alistipes Shahii

Alisitipes phage SZW_AS01, a novel lytic phage that specifically infects Alistipes shahii, was isolated from wastewater samples in Shenzhen, China. The phage's genome consists of 45,392 bp, with a GC content of 47%. The genome encodes 56 putative open reading frames (ORFs) and 1 tRNA gene. Direct terminal repeats with a length of 55 bp are present at both ends of the genome. Phylogenetic analysis of the amino acid sequences of terminase large subunit shows that phage SZW_AS01 forms a distinct branch from the Siphoviridae family phages, but is far from the Podoviridae and Myoviridae family phages. Transmission electron microscopy confirmed that SZW_AS01 belongs to the Siphoviridae family. To the best of our knowledge, this is the first report of a lytic phage infecting bacteria in the Alistipes genus.

Genome Sequence and Characterization of Five Bacteriophages Infecting Streptomyces coelicolor and Streptomyces venezuelae: Alderaan, Coruscant, Dagobah, Endor1 and Endor2

Streptomyces are well-known antibiotic producers, also characterized by a complex morphological differentiation. Streptomyces, like all bacteria, are confronted with the constant threat of phage predation, which in turn shapes bacterial evolution. However, despite significant sequencing efforts recently, relatively few phages infecting Streptomyces have been characterized compared to other genera. Here, we present the isolation and characterization of five novel Streptomyces phages. All five phages belong to the Siphoviridae family, based on their morphology as determined by transmission electron microscopy. Genome sequencing and life style predictions suggested that four of them were temperate phages, while one had a lytic lifestyle. Moreover, one of the newly sequenced phages shows very little homology to already described phages, highlighting the still largely untapped viral diversity. Altogether, this study expands the number of characterized phages of Streptomyces and sheds light on phage evolution and phage-host dynamics in Streptomyces.

Isolation and Characterization of a Novel Phage for Controlling Multidrug-Resistant Klebsiella pneumoniae

The emergence of multidrug-resistant bacterial pathogens has severely threatened global health. A phage with the ability to efficiently and specifically lyse bacteria is considered an alternative for controlling multidrug-resistant bacterial pathogens. The discovery of novel agents for controlling the infections caused by K. pneumoniae is urgent due to the broad multidrug-resistance of K. pneumoniae. Only a few phage isolates have been reported to infect multidrug-resistant K. pneumoniae. In this study, by using the multidrug-resistant K. pneumoniae strain as an indicator, a novel phage called vB_KleS-HSE3, which maintains high antibacterial activity and high physical stability, was isolated from hospital sewage. This phage infected one of four tested multidrug-resistant K. pneumoniae strains. This phage belongs to the Siphoviridae family and a comparative genomic analysis showed that this phage is part of a novel phage lineage among the Siphoviridae family of phages that infect strains of Klebsiella. Based on its features, the vB_KleS-HSE3 phage has potential for controlling infections caused by multidrug-resistant K. pneumoniae.

ISOLASI, EFEKTIVITAS, DAN KARAKTERISASI BAKTERIOFAG LITIK Salmonella enterica SEBAGAI BIOKONTROL PENYAKIT GASTROENTERITIS

Salmonella enterica merupakan salah satu bakteri patogen penyebab gastroenteritis yang ditransmisikan melalui air dan makanan terkontaminasi yang sering terjadi pada negara berkembang. Beberapa strain Salmonella enterica multi-resisten terhadap berbagai antibiotika. Bakteriofag litik pada famili Siphoviridae dapat menjadi solusi alternatif dalam mengurangi kejadian gastroenteritis oleh Salmonella enterica. Tujuan penelitian ini adalah untuk mengisolasi, mengetahui kemampuan bakteriofag litik Lytic Bacteriophage 1 (LB1) dalam melisis inangnya yaitu Salmonella enterica penyebab gastroenteritis, serta mengetahui karakterisasi bakteriofag litik LB 1 sebagai biokontrol penyakit gastroenteritis. Bakteriofag litik diisolasi dari pembuangan limbah domestik menggunakan teknik double layer plaque. Bakteriofag litik diidentifikasi berdasarkan morfologi plak, struktur litik, inang, aktivitas lisis sel bakteri Salmonella enterica, stabilitas dalam kondisi buffer yang berbeda dan karakterisasi protein. Bakteriofag litik LB1 hanya menginfeksi sel Salmonella enterica. Hasil pemeriksaan dengan menggunakan Transmission Electron Microscope (TEM), bakteriofag litik LB1 termasuk ke dalam famili Siphoviridae. Morfologi kepala hexagonal-icosahedral berdiameter 72.7 nm, dengan ekor non-kontraktil berdiameter 17.3 nm dan panjang 100 nm. Bakteriofag litik LB1 memiliki stabilitas terbaik dalam buffer Ringers suhu 4 oC yang ditunjukkan dengan penurunan plak sebesar 28% setelah 3 minggu penyimpanan. Hasil pengujian efektivitas menunjukkan bahwa bakteriofag litik LB1 dapat mengurangi populasi sel Salmonella sebanyak 67,12% setelah 8 jam inkubasi. Bakteriofag litik LB1 memiliki 8 protein yang berbeda dengan berat molekul yang beragam 11.4 kDa, 19.6 kDa, 23 kDa, 33 kDa, 58.3 kDa, 77 kDa, 94.5 kDa, 133 kDa. Studi ini menunjukkan bahwa bakteriofag litik LB1 yang diisolasi pembuangan limbah domestik dapat secara efektif mengurangi Salmonella enterica dengan cara melisis sel bakteri. Bakteriofag litik LB1 berpeluang dapat digunakan sebagai biokontrol penyakit gastroenteritis yang disebabkan oleh Salmonella enterica. Stabilitas terbaik bakteriofag litik LB1 pada penyimpanan dalam buffer Ringer di suhu dingin (4oC), memiliki karakterisasi famili Siphoviridae, dapat mengurangi Salmonella enterica sebanyak 67.12% setelah 8 jam inkubasi, dan memiliki berat molekul 11.4-133 kDa. Salmonella enterica is one of pathogenic bacteria causing gastroenteritis transmitted by water and food contamination which commonly occur in developing country. Some study reported that Salmonella serovar enterica strains were multi-resistant to various of antibiotics. Lytic bacteriophage in Siphoviridae family offered a good solution to reduce gastroenterytis disease caused by Salmonella enterica. This reseach aim was to isolate, effectivity test of LB 1 and to characterize lytic bacteriophage as biocontrol of gastroenterytis. Methodology and results were LB1 lytic bacteriophage was isolated from domestic waste using double layer plaque technique, was determined by the plaque morphology, the structure, the host range, the activity to lyse bacterial host cells, the stability of phage on different buffer conditions, and the protein characterization. The results showed that LB1 only infects Salmonella enterica. Based on Electron Microscope Observation showed that LB1 is grouped into Siphoviridae. It has hexagonal-icosahedral head with 72.7 nm in diameter and long-non contractile tail with 100 nm in diameter. LB1 had a good storage stability in Ringers buffer at low temperature (40C), with viability of bacteriophage decreased by 28% after 3 weeks of storage. The effectiveness showed that LB1 could reduce Salmonella enterica by 67.12% after 8 hours of incubation. LB 1 has different proteins with molecular weights: 11.4 kDa, 19.6 kDa, 23 kDa, 33 kDa, 58.3 kDa, 77 kDa, 94.5 kDa, and 133 kDa. The conclusion was LB1 was isolated from sewage water were identified to reduces Salmonella enterica effectively with concentration of 8.2x108 CFU/mL. LB 1 can be used as a biocontrol of gastroenterytis caused by Salmonella enterica, LB 1 has the best stability in buffer ringers in cold temperatures (4oC) and proven as Siphoviridae family, reduced Salmonella enterica by 67.12% after 8 hours of incubation, and has protein molecule with molecular weight 11.4 to 133 kDa.

Bacillus Phage vB_BtS_B83 Previously Designated as a Plasmid May Represent a New Siphoviridae Genus

The Bacillus cereus group of bacteria includes, inter alia, the species known to be associated with human diseases and food poisoning. Here, we describe the Bacillus phage vB_BtS_B83 (abbreviated as B83) infecting the species of this group. Transmission electron microscopy (TEM) micrographs indicate that B83 belongs to the Siphoviridae family. B83 is a temperate phage using an arbitrium system for the regulation of the lysis–lysogeny switch, and is probably capable of forming a circular plasmid prophage. Comparative analysis shows that it has been previously sequenced, but was mistaken for a plasmid. B83 shares common genome organization and >46% of proteins with other the Bacillus phage, BMBtp14. Phylograms constructed using large terminase subunits and a pan-genome presence–absence matrix show that these phages form a clade distinct from the closest viruses. Based on the above, we propose the creation of a new genus named Bembunaquatrovirus that includes B83 and BMBtp14.