Putative Open Reading Frames Research Articles

Genomic characterization and survey of a second luteovirus infecting blueberries

New and emerging viral problems may be contributing to blueberry decline. In this research we described a new virus detected in Oregon blueberry production field and surveyed the region for its potential spread. The complete genome sequence of a putative new member of the genus Luteovirus was obtained from blueberry (Vaccinium corymbosum L.) by high throughput sequencing and 5’/3’-RACE. The new virus was tentatively named blueberry virus M (BlVM). Its genome is 5,018 nt long with four putative open reading frames. Similarly to some recently discovered luteoviruses, BlVM does not possess any movement protein (MP). Phylogenetic analysis confirmed clustering of BlVM with the group of non-MP luteoviruses, showing blueberry virus L (BlVL) as the most similar species. Through a small-scale high throughput sequencing survey we obtained 14 additional near complete genomic sequences. A larger survey of 2,654 samples by RT-PCR in Oregon and Washington (USA) found 52 BlVM-positive plants collected from four locations in Oregon. These findings will facilitate monitoring virus distribution and assessment of potential disease associated with this new and emerging blueberry virus.

Molecular characterization of a novel fusarivirus infecting the plant-pathogenic fungus Nigrospora sphaerica.

Here, we describe a novel mycovirus, tentatively designated as "Nigrospora sphaerica fusarivirus 2" (NsFV2), which was isolated from the phytopathogenic fungus Nigrospora sphaerica strain HNXX-Ns20. NsFV2 has a single-stranded positive-sense RNA (+ ssRNA) genome of 6,156 nucleotides, excluding the poly(A) tail, and contains two putative open reading frames (ORFs). ORF1 encodes a large polypeptide of 1,509 amino acids (aa) containing a conserved RNA-dependent RNA polymerase (RdRp) domain and a viral helicase domain. The ORF1-encoded polypeptide shares 29.40-68.48% sequence identity with other fusariviruses and shares the highest sequence identity (68.48%) with Nigrospora sphaerica fusarivirus 1 (NsFV1). The small ORF2 encodes a polypeptide of 483 aa that contains a conserved chromosome segregation ATPase (Smc) domain. Multiple sequence alignments and phylogenetic analysis based on the ORF1-encoded polypeptide indicated that NsFV2 should be considered a new member of the genus Alphafusarivirus of the family Fusariviridae.

Characterization and genomic analysis phage PmP19, a novel Proteus mirabilis phage with a broad host range.

A lytic Proteus mirabilis phage, PmP19, was isolated from sewage on a farm. PmP19 has an icosahedral head (60 ± 3 nm in diameter) and a short tail (15 ± 2 nm long). Its genome, a linear, double-stranded DNA molecule 44,305 bp in length with an average GC content of 51.93%, has 52 putative open reading frames (ORFs). BLASTn comparisons and phylogenetic analysis revealed a close relationship between Pmp19 and Klebsiella phage vB_KpnP_ZK1. Bioinformatic analysis revealed that PmP19 belongs to the phage subfamily Molineuxvirinae.

Characterization and functional insights of the novel RC-type plasmid pAnox1 from Anoxybacillus gonensis 05S15

The plasmid pAnox1, isolated from Anoxybacillus gonensis 05S15, was sequenced and characterized as a circular, double-stranded DNA molecule of 1592 base pairs with a GC content of 40.01 %. Despite its cryptic nature and small genome, bioinformatic analyses identified conserved motifs associated with replication-related proteins, though BLAST searches revealed no significant homology with other plasmids. The plasmid genome contains five putative Open Reading Frames (ORFs), four palindromic sequences, and two direct repeats on both strands, suggesting regulatory roles. Electron microscopy and Southern hybridization studies confirmed that pAnox1 follows a Rolling Circle (RC) replication mode. The study further demonstrated that the plasmid encodes three distinct transcripts: ORF-1 and ORF-3 are oriented in the same direction, while ORF-5 is on the opposite strand. RACE and LACE analyses revealed transcript lengths of 903 bp for ORF1, 499 bp for ORF3, and 211 bp for ORF5. Quantitative real-time PCR estimated the relative copy number of pAnox1 at 127 ± 2 copies per chromosomal equivalent. This novel RC-type plasmid in the Anoxybacillus genome holds promise as a cloning and expression vector for biotechnological applications and in vivo protein engineering.

Biological and genome characteristics of a novel broad host-range phage ΦEP1 infecting enteroinvasive Escherichia coli

We analyzed the biological and genome characteristics of a phage infecting enteroinvasive Escherichia coli (EIEC), aiming to provide resources and a reference for the prevention and treatment of EIEC. With the EIEC preserved in our laboratory as the host bacterium, one strain of phage was isolated from the effluent sample from a chicken farm in Huzhou, Zhejiang and named ΦEP1. The titer, optimal multiplicity of infection, one-step growth curve, temperature, pH value, chloroform and bile salt sensitivity of ΦEP1 were determined by the double-layer agar plate method. The morphology of the phage was observed by transmission electron microscopy. The biocontrol effects of ΦEP1 in different food matrixes and the protective effect of this phage on Caco-2 cells were tested. The phage ΦEP1 showed the optimal multiplicity of infection of 0.1, the titer of 1.3×1010 PFU/mL, strong tolerance to temperature, pH, chloroform, and bile salt, and a broad host spectrum. Furthermore, it expressed lysis activity against multiple strains of multiple antibiotic-resistant pathogenic E. coli and Shigella with different serotypes. Phage ΦEP1 had an incubation period of 10 min, an outbreak period of 80 min, and an outbreak volume of 48 PFU/cell. According to the morphology observed by transmission electron microscopy, phage ΦEP1 belonged to the order of Caudovirales, and it had a good protective effect on Caco-2 cells. Phage ΦEP1 had a genome of 87 182 bp with the GC content of 39.80%, 128 putative open reading frames, and no antibiotic resistance genes or virulence genes. ΦEP1 inhibited the growth of EIEC in artificially contaminated milk and beef and eliminated EIEC in cell protection experiments. It significantly increased the survival rate of Caco-2 cells and down-regulated the expression of interleukin (IL)-6 and IL-1β to reduce inflammation. We obtained an EIEC-targeting phage ΦEP1 with a high titer and strong tolerance to the environment, which provided a basis for the application of phages in food preservation and other fields.

Confirmation of the gene expression patterns of viral genes within diatom-infecting DNA viruses in host diatom cells

Marine diatom-infecting DNA/RNA viruses (DI DNA/RNAVs) are of broad interest in basic and applied research on diatoms. However, the mechanisms that underly viral gene expression and its regulation in host diatoms have not been fully elucidated. In silico analysis of DI DNAV genomes revealed the presence of four putative open reading frames (ORFs), including the replication-associated protein (VP3) gene, the structural protein (VP2) gene, and genes of unknown function (the VP1 gene and the VP4 gene). In this study, we analyzed the expression of the viral ORF genes in three combinations of DI DNAVs and host diatoms. RT–PCR analysis was performed to determine the temporal expression pattern of the viral ORF genes in infected host diatoms. Compared with the VP2 and VP3 genes, the VP1 and VP4 genes exhibited increased levels of expression after infection. Furthermore, we characterized the promoter activities of the putative promoter regions in the four ORF genes. The activity of the promoter derived from the VP4 gene might be the highest among these genes. The consensus sequences among the potential promoter regions of the ORF genes were analyzed using consensus motif-finding algorithms and multiple sequence alignment programs. Some consensus sequences were found among the potential promoter regions of the ORF genes. The expression of the VP4 gene induced by strong promoter activity in the early stage of infection may be important for the viral life cycle. These findings may help researchers determine the mechanism of DI DNA/RNAVs gene expression in host diatoms and during viral infection.

Characterization of Caulimovirid-like Sequences from Upland Cotton (Gossypium hirsutum L.) Exhibiting Terminal Abortion in Georgia, USA.

In this study, we investigated the potential involvement of endogenous viral elements (EVEs) in the development of apical tissue necrosis, resulting in the terminal abortion of upland cotton (Gossypium hirsutum L.) in Georgia. The high-throughput sequence analysis of symptomatic and asymptomatic plant tissue samples revealed near-complete EVE-Georgia (EVE-GA) sequences closely related to caulimoviruses. The analysis of EVE-GA's putative open reading frames (ORFs) compared to cotton virus A and endogenous cotton pararetroviral elements (eCPRVE) revealed their similarity in putative ORFs 1-4. However, in the ORF 5 and ORF 6 encoding putative coat protein and reverse transcriptase, respectively, the sequences from EVE-GA have stop codons similar to eCPRVE sequences from Mississippi. In silico mining of the cotton genome database using EVE-GA as a query uncovered near-complete viral sequence insertions in the genomes of G. hirsutum species (~7 kb) but partial in G. tomentosum (~5.3 kb) and G. mustelinum (~5.1 kb) species. Furthermore, cotton EVEs' episomal forms and messenger RNA (mRNA) transcripts were detected in both symptomatic and asymptomatic plants collected from cotton fields. No significant yield difference was observed between symptomatic and asymptomatic plants of the two varieties evaluated in the experimental plot. Additionally, EVEs were also detected in cotton seeds and seedlings. This study emphasizes the need for future research on EVE sequences, their coding capacity, and any potential role in host immunity or pathogenicity.

Detection and Genomic Characterization of Torque Teno Virus in Pneumoconiosis Patients in China.

Pneumoconiosis is a common occupational disease that can worsen with accompanying infection. Torque teno virus (TTV) is a prevalent human virus with multiple genotypes that can chronically and persistently infect individuals. However, the prevalence of TTV in pneumoconiosis patients is still unclear. This research aims to detect the presence and prevalence of TTV in the alveolar lavage fluid of pneumoconiosis patients in the Hunan Province of China using PCR. As a result, a 65.5% positive rate (19 out of 29) of TTV was detected. The TTV detection rate varies among different stages of silicosis and different pneumoconiosis patient ages. Nine novel TTV genomes ranging in size from 3719 to 3908 nt, named TTV HNPP1, HNPP2, HNPP3, HNPP4, HNPP5, HNPP6-1, HNPP6-2, HNPP7-1 and HNPP7-2, were identified. A genomic comparison and phylogenetic analysis indicated that these nine TTVs represent five different species with high genetic diversity which belong to the genus Alphatorquevirus. HNPP6-1 and HNPP6-2 belong to TTV3, HNPP5 belongs to TTV13, HNPP1 belongs to TTV24, HNPP4 belongs to TTV20, and the others belong to TTV19. The genomes of TTV HNPP1, HNPP6-1, and HNPP6-2 contain three putative open reading frames (ORFs) coding for proteins, ORF1, ORF2, and ORF3, while the other six TTV genomes contain two ORFs coding for proteins, ORF1 and ORF2. These results provide the first description of TTV epidemiology in pneumoconiosis patients in China. The newly identified TTV genome sequences reveal the high genetic diversity of TTV in pneumoconiosis patients and could contribute to a deeper understanding of TTV retention and infection in humans.

Characterization of an Escherichia coli phage Tequatrovirus YZ2 and its application in bacterial wound infection

The increasing prevalence of drug-resistant Escherichia coli (E. coli) resulting from the excessive utilization of antibiotics necessitates the immediate exploration of alternative approaches to counteract pathogenic E. coli. Phages, with their unique antibacterial mechanisms, are considered promising candidates for treating bacterial infections. Herein, we isolated a lytic Escherichia phage Tequatrovirus YZ2 (phage YZ2), which belongs to the genus Tequatrovirus. The genome of phage YZ2 consists of 168,356 base pairs with a G + C content of 35.34% and 269 putative open reading frames (ORFs). Of these, 146 ORFs have been annotated as functional proteins associated with nucleotide metabolism, structure, transcription, DNA replication, translation, and lysis. In the mouse model of a skin wound infected by E. coli, phage YZ2 therapy significantly promoted the wound healing. Furthermore, histopathological analysis revealed reductions in IL-1β and TNF-α and increased VEGF levels, indicating the potential of phages as effective antimicrobial agents against E. coli infection.

Capture of armA by a novel ISCR element, ISCR28

ISCR28 is a fully functional and active member of the IS91-like family of insertion sequences. ISCR28 is 1,708-bp long and contains a 1,293-bp long putative open reading frame that codes a transposase. Sixty ISCR28-containing sequences from GenBank generated 27 non-repeat genetic contexts, all of which represented naturally occurring biological events that had occurred in a wide range of gram-negative organisms. Insertion of ISCR28 into target DNA preferred the presence of a 5′-GXXT-3′ sequence at its terIS (replication terminator) end. Loss of the first 4 bp of its oriIS (origin of replication) likely caused ISCR28 to be trapped in ISApl1-based transposons or similar structures. Loss of terIS and fusion with a mobile element upstream likely promoted co-transfer of ISCR28 and the downstream resistance genes. ArmA and its downstream intact ISCR28 can be excised from recombinant pKD46 plasmids forming circular intermediates, further elucidating its activity as a transposase.

A circular single-stranded DNA mycovirus infects plants and confers broad-spectrum fungal resistance

Circular single-stranded DNA (ssDNA) viruses have been rarely found in fungi, and the evolutionary and ecological relationships among ssDNA viruses infecting fungi and other organisms remain unclear. In this study, a novel circular ssDNA virus, tentatively named Diaporthe sojae circular DNA virus 1 (DsCDV1), was identified in the phytopathogenic fungus Diaporthe sojae isolated from pear trees. DsCDV1 has a monopartite genome (3185 nt in size) encapsidated in isometric virions (21–26 nm in diameter). The genome comprises seven putative open reading frames encoding a discrete replicase (Rep) split by an intergenic region, a putative capsid protein (CP), several proteins of unknown function (P1–P4), and a long intergenic region. Notably, the two split parts of DsCDV1 Rep share high identities with the Reps of Geminiviridae and Genomoviridae, respectively, indicating an evolutionary linkage with both families. Phylogenetic analysis based on Rep or CP sequences placed DsCDV1 in a unique cluster, supporting the establishment of a new family, tentatively named Gegemycoviridae, intermediate to both families. DsCDV1 significantly attenuates fungal growth and nearly erases fungal virulence when transfected into the host fungus. Remarkably, DsCDV1 can systematically infect tobacco and pear seedlings, providing broad-spectrum resistance to fungal diseases. Subcellular localization analysis revealed that DsCDV1 P3 is systematically localized in the plasmodesmata, while its expression in trans-complementation experiments could restore systematic infection of a movement-deficient plant virus, suggesting that P3 is a movement protein. DsCDV1 exhibits unique molecular and biological traits not observed in other ssDNA viruses, serving as a link between fungal and plant ssDNA viruses and presenting an evolutionary connection between ssDNA viruses and fungi. These findings contribute to expanding our understanding of ssDNA virus diversity and evolution, offering potential biocontrol applications for managing crucial plant diseases.

Genome assembly and annotation of Spilosoma obliqua multicapsid nucleopolyhedrovirus from Bihar hairy caterpillar, an agriculturally important insect pest

AbstractSpilosoma obliqua nucleopolyhedrovirus (SpobNPV) is known as a biocontrol agent against S. obliqua a polyphagous insect. Genome of an isolate designated as SpobMNPV was sequenced and found to have 136,141 bp, 139 putative open reading frames (ORFs) on both sense (48%) and anti‐sense (52%) strands and 97.91% nucleotide similarity with Hyphantria cunea nucleopolyhedrovirus (HycuNPV). All the 38 core genes of baculoviruses were identified and validated in the SpobMNPV genome, which differed from SpobNPV–Manipur isolate in several aspects. In SpobMNPV genome, 7 h were found comprised of 2 to 16 repeated units of 67‐bp at each site with an imperfect 30‐bp palindrome near the centre in both orientations. Comparison of consensus palindrome sequences (hrcons) present in hrs with that of selected alphabaculovirus group I NPVs revealed them to be completely conserved at each side of the hrcons, that is 1‐GxTTTxC‐7 and 22‐TxGxAAAxC‐30. Based on phylogenetic analysis of 38 core genes, SpobMNPV was found closest to the HycuNPV in the group I alphabaculovirus. The complete genome of this isolate is being reported for the first time from North India. The information on genome analysis of SpobMNPV will be an addition to the available database on alphabaculoviruses and also accelerate research on SpobNPV as a component of integrated management of S. obliqua in many economically important crops.

Whole genome analysis, thermal and UV-tolerance of Lactococcus phage BIM BV-114 isolated from cheese brine

Lactococcus phages that belong to the genus Ceduovirus are among the three most frequently isolated phage groups infecting Lactococcus lactis starter strains in dairy plants. In this study, we characterized virulent Lactococcus phage BIM BV-114 isolated from industrial cheese brine in Belarus and identified as Ceduovirus. The bacteriophage demonstrated a relatively short lytic cycle (latent period of 23 ± 5 min, lysis time of 90 ± 5 min), high thermal stability (inactivation after 7 min at 95 °C in skimmed milk) and tolerance to UV radiation (inactivation time – 15 min), indicating adaptation for better persistence in dairy facilities. The genome of the phage BIM BV-114 (21 499 bp; 37 putative open reading frames) has a similar organization to that of other Ceduovirus phages. RLf1_00140 and RLf_00050 gene products, found in the early genes region, may be involved in the sensitivity of phage to the lactococcal abortive infection mechanisms AbiV and AbiQ, respectively. Furthermore, nucleotide deletion, observed in the middle region of the gene encoding putative tape measure protein (RLf1_00300), is possibly responsible for increased thermal tolerance of phage BIM BV-114. Together, these findings will contribute to a better knowledge of virulent Lactococcus phages and the development of effective methods of their control for dairy technologies.

Identification and genomic analysis of a novel temperate bacteriophage infecting Labrenzia aggregata isolated from the Mariana Trench

In marine environments, viruses play a pivotal role, yet deep-sea bacteriophages remains largely uncharacterized. The bacterium Labrenzia aggregata RF14, isolated from the Mariana Trench at a depth of 4,000 meters, harbors prophage regions based on a previous study. In this study, we induced a temperate bacteriophage from it using mitomycin C. The bacteriophage exhibited an icosahedral structure with a non-extendable tail and was named vB_LagS-V1. The genome size of it is 39,329 bps with a 59.46% G+C content, encoding 60 putative open reading frames. Genomic and phylogenetic analyses demonstrated that vB_LagS-V1 along with many bacteriophages infecting Hyphomicrobiales, constituted a newly unclassified family, which we designated as Hyphoviridae. Within this novel family, vB_LagS-V1 is distinct with isolated phages and clustered with two uncultured prophages within Labrenzia, forming an unclassified new genus, given a name of Labrenmarinevirus. The codon usage correlation and absence of tRNAs found in vB_LagS-V1, also prevail in some deep-sea bacteriophages, highlighting their adaptations to the deep-sea prokaryotic hosts. Moreover, vB_LagS-V1 encoded two auxiliary metabolic genes, cysteine dioxygenase and phosphoadenosine phosphosulfate reductase, which might help the phage and its host adapt to high hydrostatic pressure in the deep-sea environments. Our study will significantly contribute to the understanding of deep-sea bacteriophages and their interactions with hosts in extreme environments.

Analysis of unintended open reading frame ORF-130 expression in maize event MZIR098 by LC-MS as part of the allergenicity risk assessment of genetically modified crops

Rigorous safety assessments are required for genetically modified (GM) crops to support their use as food or feed. These assessments include bioinformatics analysis of the amino acid sequences of all potential open reading frames (ORFs) encoded in the exogenous DNA introduced into the plant through genetic modification. Sequence similarities of potential DNA products to known or potential toxins and allergens trigger subsequent assessments of potential hazards for the consumer or the environment. The bioinformatic analysis of Syngenta maize event MZIR098 insert DNA revealed a total of 415 ORFs. One putative ORF (ORF-130) triggered further risk assessment based on its sequence similarities with known allergens, including peanut allergen Ara h 1, wheat glutenin, and others. We examined the potential expression of ORF-130 at a protein level to exclude the possibility of exposure during consumption of MZIR098 maize. Specifically, we developed and validated a liquid chromatography approach coupled to a tandem mass spectrometry (LC-MS/MS) method, called parallel reaction monitoring (PRM). This measure of expression was quantitative and targeted against the peptides unique to ORF-130. The linear range of the method was 25-5000 amol ORF-130/µl. Both, limit of detection (LOD) and lower limit of quantitation (LLOQ) of the method, were 12 μg ORF-130 per gram of dry MZIR098 maize leaf. The method was accurate and specific to ORF-130. PRM analysis of MZIR098 extracts did not detect putative ORF-130 above the limits of detection/quantitation. Furthermore, the broad proteomic data dependent acquisition (DDA) analysis of the MZIR098 extracts demonstrated no evidence of ORF-130 corroborating the PRM analysis. Our results confirm that ORF-130 is not characterized as a functional gene in Syngenta maize event MZIR098 insert DNA and does not express gene products at detectable levels.

Folded Alpha Helical Putative New Proteins from Apilactobacillus kunkeei

The emergence of new proteins is a central question in biology. Most tertiary protein folds known to date appear to have an ancient origin, but it is clear from bioinformatic analyses that new proteins continuously emerge in all organismal groups. However, there is a paucity of experimental data on new proteins regarding their structure and biophysical properties. We performed a detailed phylogenetic analysis and identified 48 putative open reading frames in the honeybee-associated bacterium Apilactobacillus kunkeei for which no or few homologs could be identified in closely-related species, suggesting that they could be relatively new on an evolutionary time scale and represent recently evolved proteins. Using circular dichroism-, fluorescence- and nuclear magnetic resonance (NMR) spectroscopy we investigated six of these proteins and show that they are not intrinsically disordered, but populate alpha-helical dominated folded states with relatively low thermodynamic stability (0–3 kcal/mol). The NMR and biophysical data demonstrate that small new proteins readily adopt simple folded conformations suggesting that more complex tertiary structures can be continuously re-invented during evolution by fusion of such simple secondary structure elements. These findings have implications for the general view on protein evolution, where de novo emergence of folded proteins may be a common event.

Molecular characterization of a novel narnavirus infecting the phytopathogenic fungus Botryosphaeria dothidea.

Here, a novel mycovirus, Botryosphaeria dothidea narnavirus 5 (BdNV5), was discovered in the plant-pathogenic fungus Botryosphaeria dothidea strain ZM210167-1. The BdNV5 genome sequence is 2,397 nucleotides (nt) in length and contains a putative open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp) with a molecular mass of 72.77 kDa. A BLASTp search using the RdRp amino acid (aa) sequence showed that it was most similar to the RdRp of Botryosphaeria dothidea narnavirus 4 (42.35%). In a phylogenetic tree based on RdRp aa sequences, BdNV5 clustered with members of the family Narnaviridae. BdNV5 is thus a novel member of the family Narnaviridae infecting the phytopathogenic fungus B. dothidea.

Genomic characterization of SNW-1, a novel prophage of the deep-sea vent chemolithoautotroph Sulfurimonas indica NW79.

The globally widespread genus Sulfurimonas are playing important roles in different habitats, including the deep-sea hydrothermal vents. However, phages infecting Sulfurimonas have never been isolated and characterized to date. In the present study, a novel prophage SNW-1 was identified from Sulfurimonas indica NW79. Whole genome sequencing resulted in a circular, double-stranded DNA molecule of 37,096 bp with a mol% G+C content of 37. The genome includes 64 putative open reading frames, 33 of which code for proteins with predicted functions. Presence of hallmark genes associated with Caudoviricetes and genes involved in lysis and lysogeny indicated that SNW-1 should be a temperate, tailed phage. Phylogenetic and comparative proteomic analyses suggested that Sulfurimonas phage SNW-1 was distinct from other double stranded DNA phages and might represent a new viral genus.

Co-Occurrence of tet(X4) and blaNDM-5 in Escherichia coli Isolates of Inpatient Origin in Guangzhou, China.

Tigecycline, one of the last-resort therapeutic options for complicated infections caused by multidrug-resistant pathogens, especially carbapenem-resistant Enterobacterales and Acinetobacter in recent years. The emergence of antibiotic-resistant bacteria and antibiotic-resistant genes has threatened the effectiveness of antibiotics and public health with the excessive use of antibiotics in clinics. However, the emergence and dissemination of high-level mobile tigecycline-resistance gene tet(X) is challenging for clinical effectiveness of antimicrobial agent. This study aimed to characterize an E. coli strain T43, isolated from an inpatient in a teaching hospital in China. The E. coli T43 was resistant to almost all antimicrobials except colistin and consisted of a 4,774,080 bp chromosome and three plasmids. Plasmids pT43-1 and pT43-2 contained tigecycline-resistance gene tet(X4). Plasmid pT43-1 had a size of 152,423 bp with 51.05% GC content and harbored 151 putative open reading frames. pT43-1 was the largest plasmid in strain T43 and carried numerous resistance genes, especially tigecycline resistance gene tet(X4) and carbapenemase resistance gene blaNDM-5. The tet(X) gene was associated with IS26. Co-occurrence of numerous resistance genes in a single plasmid possibly contributed to the dissemination of these genes under antibiotics stress. It might explain the presence of clinically crucial resistance genes tet(X) and blaNDM-5 in clinics. This study suggested the applicable use of antibiotics and continued surveillance of tet(X) and blaNDM-5 in clinics are imperative.

Characterizations of novel broad-spectrum lytic bacteriophages Sfin-2 and Sfin-6 infecting MDR Shigella spp. with their application on raw chicken to reduce the Shigella load

The evidence and prevalence of multidrug-resistant (MDR) Shigella spp. poses a serious global threat to public health and the economy. Food- or water-borne MDR Shigella spp. demands an alternate strategy to counteract this threat. In this regard, phage therapy has garnered great interest from medical practitioners and researchers as a potential way to combat MDR pathogens. In this observation, we isolated Shigella phages from environmental water samples and tested against various clinically isolated MDR Shigella spp. In this study, we have defined the isolation and detailed physical and genomic characterizations of two phages Sfin-2 and Sfin-6 from environmental water samples. The phages exhibited potent lytic activity against Shigella flexneri, Shigella dysenteriae, and Shigella sonnei. They showed absorption within 5–10 min, a burst size ranging from ~74 to 265 PFU/cell, and a latent period of 5–20 min. The phages were stable at a broad pH range and survived an hour at 50°C. The purified phages Sfin-2 and Sfin-6 belong to the Siphoviridae family with an isometric head (64.90 ± 2.04 nm and 62.42 ± 4.04 nm, respectively) and a non-contractile tail (145 ± 8.5 nm and 148.47 ± 14.5 nm, respectively). The in silico analysis concluded that the size of the genomic DNA of the Sfin-2 phage is 50,390 bp with a GC content of 44.90%, while the genome size of the Sfin-6 phage is 50,523 bp with a GC content of 48.30%. A total of 85 and 83 putative open reading frames (ORFs) were predicted in the Sfin-2 and Sfin-6 phages, respectively. Furthermore, a comparative genomic and phylogenetic analysis revealed that both phages represented different isolates and novel members of the T1-like phages. Sfin-2 and Sfin-6 phages, either individually or in a cocktail form, showed a significant reduction in the viable Shigella count on raw chicken samples after 72 h of incubation. Therefore, these results indicate that these phages might have a potential role in therapeutic approaches designed for shigellosis patients as well as in the biological control of MDR Shigella spp. in the poultry or food industry during the course of meat storage.