A genome-wide cytotoxicity screen of cluster F1 mycobacteriophage Girr reveals novel inhibitors of Mycobacterium smegmatis growth.

Over the past decade, thousands of bacteriophage genomes have been sequenced and annotated. A striking observation from this work is that known structural features and functions cannot be assigned for >65% of the encoded proteins. One approach to begin experimentally elucidating the function of these uncharacterized gene products is genome-wide screening to identify phage genes that confer phenotypes of interest like inhibition of host growth. This study describes the results of a screen evaluating the effects of overexpressing each gene encoded by the temperate Cluster F1 mycobacteriophage Girr on the growth of the host bacterium Mycobacterium smegmatis. Overexpression of 29 of the 102 Girr genes (~28% of the genome) resulted in mild to severe cytotoxicity. Of the 29 toxic genes described, 12 have no known function and are predominately small proteins of <125 amino acids. Overexpression of the majority of these 12 cytotoxic no known functions proteins resulted in moderate to severe growth reduction and represent novel antimicrobial products. The remaining 17 toxic genes have predicted functions, encoding products involved in phage structure, DNA replication/modification, DNA binding/gene regulation, or other enzymatic activity. Comparison of this dataset with prior genome-wide cytotoxicity screens of mycobacteriophages Waterfoul and Hammy reveals some common functional themes, though several of the predicted Girr functions associated with cytotoxicity in our report, including genes involved in lysogeny, have not been described previously. This study, completed as part of the HHMI-supported SEA-GENES project, highlights the power of parallel, genome-wide overexpression screens to identify novel interactions between phages and their hosts.

Complete genome sequences and characteristics of mycobacteriophages Diminimus, Dulcita, Glaske16, and Koreni.

Complete genome sequences of four novel mycobacteriophages, Diminimus, Dulcita, Glaske16, and Koreni, isolated from soil are presented. All these bacteriophages belong to subcluster M1, except Koreni that belongs to subcluster A4. Moreover, all have siphovirus morphologies, with genome sizes ranging from 51,055 to 81,156 bp.

Phage engineering for development of diagnostic tools.

The bacteriophages rely on the host cell to provide energy and resources for their own replication. Antibody-based diagnostic tests rely on the antibody and the biomarker interactions. Since, most of these diagnostic tools employ the use of antibodies; hence, they require intensive storage protocols at cold conditions and incur high time and capital cost due to their production and purification process. Phage-based diagnostics can overcome this limitation. Bacteriophages, have been used as emerging tools for the detection of various pathogens. Rapid phage-mediated detection assays have become commercial diagnostic tools. Conventional method and new cloning approaches have been followed to specifically detect a disease- causing microbial strains. This review discusses use of Phage typing as diagnostic tools, phage-based detection methods, and their usage for signal amplification. Design rules for reporter phage engineering are also discussed followed by different engineering platforms for phage genome editing. We also discuss recent examples of how phage research is influencing the recent advances in the development of phage-based diagnostics for ultra-sensitive detection of various bio-species, outlining the advantages and limitations of detection technology of phage-based assays.

New advances in the treatments of drug-resistant tuberculosis.

INTRODUCTION: TB is associated with high mortality and morbidity among infected individuals and a high transmission rate from person to person. Despite the availability of vaccines and several anti-TB,TB infection continues to increase. Global resistance to TB remains the greatest challenge. There has not been extensive research into a new treatment and management strategy for TB resistance therapy. This review is based on a review of new advances and alternative drugs in the treatment of drug-resistant TB. AREAS COVERED: New drug-resistant Mycobacterium tuberculosis therapy involves a combination of the latest TB drugs, new anti-TB drugs based on medicinal plant extracts for drug-resistant TB, mycobacteriophage therapy, the CRISPR/Cas9 system, and nanotechnology. EXPERT OPINION: It is necessary to determine the function of individual gene alterations in drug-resistant TB. A combination of the most recent anti-TB drugs, such as bedaquiline and delamanid, is recommended. Longitudinal studies and animal model experiments with some medicinal plant extracts are required for better results. Nanotechnology has the potential to reduce drug side effects. Useful efficacy of phage therapy and CRISPR-cas9 technology as adjunct therapies for the management of drug-resistant TB.

Experimental Studies of the Liposomal Form of Lytic Mycobacteriophage D29 for the Treatment of Tuberculosis Infection.

We have studied the antimycobacterial efficacy of the liposomal preparation of mycobacteriophage D29 on models of tuberculous granuloma in vitro and in the experiment on laboratory mice of the relatively resistant strain C57BL/6, infected with the virulent strain of M. tuberculosis H37Rv. We have shown the preparation of liposomal preparation of the lytic mycobacteriophages and its characteristics. The experiments showed a significant lytic effect of the liposomal form of mycobacteriophage D29 both on the model of tuberculous granuloma formed by human blood mononuclear cells in vitro, which is formed in the presence of Mycobacterium tuberculosis and on the model of tuberculous infection in C57BL/6 mice. Keywords: mycobacteriophage D29, M. tuberculosis, liposomes, tuberculous granuloma in vitro, tuberculosis infection and its treatment.

Intrapulmonary Treatment with Mycobacteriophage LysB Rapidly Reduces Mycobacterium abscessus Burden.

Intrinsic and acquired antibiotic resistance in Mycobacterium abscessus presents challenges in infection control, and new therapeutic strategies are needed. Bacteriophage therapy shows promise, but variabilities in M. abscessus phage susceptibility limits its broader utility. We show here that a mycobacteriophage-encoded lysin B (LysB) efficiently and rapidly kills both smooth- and rough-colony morphotype M. abscessus strains and reduces the pulmonary bacterial load in mice. LysB aerosolization presents a plausible treatment for pulmonary M. abscessus infections.

Bacteriophages of Mycobacterium tuberculosis, their diversity, and potential therapeutic uses: a review.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (M. tuberculosis) is a highly infectious disease and worldwide health problem. Based on the WHO TB report, 9 million active TB cases are emerging, leading to 2 million deaths each year. The recent emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) strains emphasizes the necessity to improve novel therapeutic plans. Among the various developing antibacterial approaches, phage therapy is thought to be a precise hopeful resolution. Mycobacteriophages are viruses that infect bacteria such as Mycobacterium spp., containing the M. tuberculosis complex. Phages and phage-derived proteins can act as promising antimicrobial agents. Also, phage cocktails can broaden the spectrum of lysis activity against bacteria. Recent researches have also shown the effective combination of antibiotics and phages to defeat the infective bacteria. There are limitations and concerns about phage therapy. For example, human immune response to phage therapy, transferring antibiotic resistance genes, emerging resistance to phages, and safety issues. So, in the present study, we introduced mycobacteriophages, their use as therapeutic agents, and their advantages and limitations as therapeutic applications.

Phylogenomic analyses and host range prediction of cluster P mycobacteriophages.

Bacteriophages, infecting bacterial hosts in every environment on our planet, are a driver of adaptive evolution in bacterial communities. At the same time, the host range of many bacteriophages-and thus one of the selective pressures acting on complex microbial systems in nature-remains poorly characterized. Here, we computationally inferred the putative host ranges of 40 cluster P mycobacteriophages, including members from 6 subclusters (P1-P6). A series of comparative genomic analyses revealed that mycobacteriophages of subcluster P1 are restricted to the Mycobacterium genus, whereas mycobacteriophages of subclusters P2-P6 are likely also able to infect other genera, several of which are commonly associated with human disease. Further genomic analysis highlighted that the majority of cluster P mycobacteriophages harbor a conserved integration-dependent immunity system, hypothesized to be the ancestral state of a genetic switch that controls the shift between lytic and lysogenic life cycles-a temperate characteristic that impedes their usage in antibacterial applications.

Incorporation of Mycobacteriophage Fulbright into Polycaprolactone Electrospun Nanofiber Wound Dressing.

The Genus Mycobacterium includes pathogens known to cause disease in mammals such as tuberculosis (Mycobacterium tuberculosis) and skin infections (M. abscessus). M. smegmatis is a model bacterium that can cause opportunistic infections in human tissues and, rarely, a respiratory disease. Due to the emergence of multidrug-resistant bacteria, phage therapy is potentially an alternative way of treating these bacterial infections. As bacteriophages are specific to their bacterial host, it ensures that the normal flora is unharmed. Fulbright is a mycobacteriophage that infects the host bacteria M. smegmatis. The main goal of this study is to incorporate Mycobacteriophage Fulbright into a polycaprolactone (PCL) nanofiber and test its antimicrobial effect against the host bacteria, M. smegmatis. Stability tests conducted over 7 days showed that the phage titer does not decrease when in contact with PCL, making it a promising vehicle for phage delivery. Antimicrobial assays showed that PCL_Fulbright effectively reduces bacterial concentration after 24 h of contact. In addition, when stored at -20 °C, the phage remains viable for up to eleven months in the fiber. Fulbright addition on the nanofibrous mats resulted in an increase in water uptake and decrease in the mechanical properties (strength and Young's modulus) of the membranes, indicating that the presence of phage Fulbright can greatly enhance the physical and mechanical properties of the PCL. Cytotoxicity assays showed that PCL_Fulbright is not cytotoxic to Balbc/3T3 mouse embryo fibroblast cell lines; thus, phage-incorporated PCL is a promising alternative to antibiotics in treating skin infections.

Phylogenetic relationships and codon usage bias amongst cluster K mycobacteriophages.

Bacteriophages infecting pathogenic hosts play an important role in medical research, not only as potential treatments for antibiotic-resistant infections but also offering novel insights into pathogen genetics and evolution. A prominent example is cluster K mycobacteriophages infecting Mycobacterium tuberculosis, a causative agent of tuberculosis in humans. However, as handling M. tuberculosis as well as other pathogens in a laboratory remains challenging, alternative nonpathogenic relatives, such as Mycobacterium smegmatis, are frequently used as surrogates to discover therapeutically relevant bacteriophages in a safer environment. Consequently, the individual host ranges of the majority of cluster K mycobacteriophages identified to date remain poorly understood. Here, we characterized the complete genome of Stinson, a temperate subcluster K1 mycobacteriophage with a siphoviral morphology. A series of comparative genomic analyses revealed strong similarities with other cluster K mycobacteriophages, including the conservation of an immunity repressor gene and a toxin/antitoxin gene pair. Patterns of codon usage bias across the cluster offered important insights into putative host ranges in nature, highlighting that although all cluster K mycobacteriophages are able to infect M. tuberculosis, they are less likely to have shared an evolutionary infection history with Mycobacterium leprae (underlying leprosy) compared to the rest of the genus' host species. Moreover, subcluster K1 mycobacteriophages are able to integrate into the genomes of Mycobacterium abscessus and Mycobacterium marinum-two bacteria causing pulmonary and cutaneous infections which are often difficult to treat due to their drug resistance.

Phage Therapy for Mycobacterium Abscessus and Strategies to Improve Outcomes.

Members of Mycobacterium abscessus complex are known for causing severe, chronic infections. Members of M. abscessus are a new "antibiotic nightmare" as one of the most resistant organisms to chemotherapeutic agents. Treatment of these infections is challenging due to the either intrinsic or acquired resistance of the M. abscessus complex to the available antibiotics. Recently, successful phage therapy with a cocktail of three phages (one natural lytic phage and two engineered phages) every 12 h for at least 32 weeks has been reported against a severe case of the disseminated M. abscessus subsp. massiliense infection, which underlines the high value of phages against drug-resistant superbugs. This report also highlighted the limitations of phage therapy, such as the absence of lytic phages with a broad host-range against all strains and subspecies of the M. abscessus complex and also the risk of phage resistant bacteria over treatment. Cutting-edge genomic technologies have facilitated the development of engineered phages for therapeutic purposes by introducing new desirable properties, changing host-range and arming the phages with additional killing genes. Here, we review the available literature and suggest new potential solutions based on the progress in phage engineering that can help to overcome the present limitations of M. abscessus treatment.

Mycobacteriophages as Potential Therapeutic Agents against Drug-Resistant Tuberculosis.

The current emergence of multi-, extensively-, extremely-, and total-drug resistant strains of Mycobacterium tuberculosis poses a major health, social, and economic threat, and stresses the need to develop new therapeutic strategies. The notion of phage therapy against bacteria has been around for more than a century and, although its implementation was abandoned after the introduction of drugs, it is now making a comeback and gaining renewed interest in Western medicine as an alternative to treat drug-resistant pathogens. Mycobacteriophages are genetically diverse viruses that specifically infect mycobacterial hosts, including members of the M. tuberculosis complex. This review describes general features of mycobacteriophages and their mechanisms of killing M. tuberculosis, as well as their advantages and limitations as therapeutic and prophylactic agents against drug-resistant M. tuberculosis strains. This review also discusses the role of human lung micro-environments in shaping the availability of mycobacteriophage receptors on the M. tuberculosis cell envelope surface, the risk of potential development of bacterial resistance to mycobacteriophages, and the interactions with the mammalian host immune system. Finally, it summarizes the knowledge gaps and defines key questions to be addressed regarding the clinical application of phage therapy for the treatment of drug-resistant tuberculosis.

Indole chalcones: Design, synthesis, in vitro and in silico evaluation against Mycobacterium tuberculosis.

Indole chalcones were designed and synthesized as a promising set of compounds against H37Rv strain of Mycobacterium tuberculosis. Within this library of compounds, (E)-1-(furan-3-yl)-3-(1H-indol-3-yl)prop-2-en-1-one (18), (E)-3-(1H-indol-3-yl)-1-(thiophen-2-yl)prop-2-en-1-one (20) and (E)-2-((1H-indol-2-yl)methylene)cyclopentan-1-one (24) displayed high anti-tubercular activity at 50 µg/ml with MIC values of 210, 197 and 236 µM respectively. The in-silico studies revealed that compound 18 exhibit binding modes similar to FAS-II inhibitors like INH or Thiolactomycin against KasA protein. Cytotoxicity assay results suggest that the compounds 18, 20 and 24 are non-cytotoxic to human megakaryocytes and murine B cells.

A rapid viability and drug-susceptibility assay utilizing mycobacteriophage as an indicator of drug susceptibilities of Anti-TB drugs against Mycobacterium smegmatis mc2 155.

Background: A rapid in-house TM4 mycobacteriophage-based assay, to identify multidrug resistance against various anti-tuberculosis drugs, using the fast-growing Mycobacterium smegmatis mc2 155 in a microtiter plate format was evaluated, based on phage viability assays. Methods: A variety of parameters were optimized before the study including the minimum incubation time for the drugs, phage and M. smegmatis mc2 155 to be in contact. An increase in phage numbers over 2 h was indicative that M. smegmatis mc2 155 is resistant to the drugs under investigation, however when phage numbers remained static, M. smegmatis mc2 155 found to be sensitive to the drug. Results: The study confirmed that the data are statistically significant and that M. smegmatis mc2 155 is, in fact, sensitive to isonazid, iifampicin, pyranzaimide, and ethambutol as phage numbers doubled over 2 h (P = 0.015, 0.018, 0.014, and 0.020). The study also confirmed that M. smegmatis mc2 155 is resistant to the drugs ampicillin, erythromycin, amoxicillin streptomycin as phage numbers remain static over the same 2 h period (P = 0.028, 0.052, 0.049, and 0.04). This drug-susceptibility profiling of eight different drugs against M. smegmatis mc2 155 was detected in as little as 1½ days with a cost of ~ one euro and fifteen cent to test four drugs. Conclusion: This test is rapid to perform and will have widespread applications in drug-susceptibility testing of other members of the mycobacterial genus. In addition, the platform could also be used as a tool for high-throughput screening of novel antimycobacterial drugs. The main assets of this assay include its relatively cheap cost, versatility, and quick turnaround time.

Directed Evolution of a Mycobacteriophage.

Bacteriophages represent an alternative strategy to combat pathogenic bacteria. Currently, Mycobacterium tuberculosis infections constitute a major public health problem due to extensive antibiotic resistance in some strains. Using a non-pathogenic species of the same genus as an experimental model, Mycobacterium smegmatis, here we have set up a basic methodology for mycobacteriophage growth and we have explored directed evolution as a tool for increasing phage infectivity and lytic activity. We demonstrate mycobacteriophage adaptation to its host under different conditions. Directed evolution could be used for the development of future phage therapy applications against mycobacteria.

Deletion of MSMEG_1350 in Mycobacterium smegmatis causes loss of epoxy-mycolic acids, fitness alteration at low temperature and resistance to a set of mycobacteriophages.

Mycobacterium smegmatis is intrinsically resistant to thiacetazone, an anti-tubercular thiourea; however we report here that it causes a mild inhibition in growth in liquid medium. Since mycolic acid biosynthesis was affected, we cloned and expressed Mycobacterium smegmatis mycolic acid methyltransferases, postulated as targets for thiacetazone in other mycobacterial species. During this analysis we identified MSMEG_1350 as the methyltransferase involved in epoxy mycolic acid synthesis since its deletion led to their total loss. Phenotypic characterization of the mutant strain showed colony morphology alterations at all temperatures, reduced growth and a slightly increased susceptibility to SDS, lipophilic and large hydrophilic drugs at 20 °C with little effect at 37 °C. No changes were detected between parental and mutant strains in biofilm formation, sliding motility or sedimentation rate. Intriguingly, we found that several mycobacteriophages severely decreased their ability to form plaques in the mutant strain. Taken together our results prove that, in spite of being a minor component of the mycolic acid pool, epoxy-mycolates are required for a proper assembly and functioning of the cell envelope. Further studies are warranted to decipher the role of epoxy-mycolates in the M. smegmatis cell envelope.

Mycobacteriophage Lysis Enzymes: Targeting the Mycobacterial Cell Envelope.

Mycobacteriophages are viruses that specifically infect mycobacteria, which ultimately culminate in host cell death. Dedicated enzymes targeting the complex mycobacterial cell envelope arrangement have been identified in mycobacteriophage genomes, thus being potential candidates as antibacterial agents. These comprise lipolytic enzymes that target the mycolic acid-containing outer membrane and peptidoglycan hydrolases responsive to the atypical mycobacterial peptidoglycan layer. In the recent years, a remarkable progress has been made, particularly on the comprehension of the mechanisms of bacteriophage lysis proteins activity and regulation. Notwithstanding, information about mycobacteriophages lysis strategies is limited and is mainly represented by the studies performed with mycobacteriophage Ms6. Since mycobacteriophages target a specific group of bacteria, which include Mycobacterium tuberculosis responsible for one of the leading causes of death worldwide, exploitation of the use of these lytic enzymes demands a special attention, as they may be an alternative to tackle multidrug resistant tuberculosis. This review focuses on the current knowledge of the function of lysis proteins encoded by mycobacteriophages and their potential applications, which may contribute to increasing the effectiveness of antimycobacterial therapy.

Beyond the Limits: tRNA Array Units in Mycobacterium Genomes.

tRNA array unit, a genomic region presenting an intriguing high tRNA gene number and density, was supposed to occur only in few bacteria phyla, particularly Firmicutes. Here, we identified and characterized an abundance and diversity of tRNA array units in Mycobacterium associated genomes. These genomes comprised chromosome, bacteriophages and plasmids from mycobacteria. Firstly, we had identified 32 tRNA genes organized in an array unit within a mycobacteria plasmid genome and therefore, we hypothesized the presence of such structures in Mycobacterium genus. However, at the time, bioinformatics tools only predict tRNA genes, not characterizing their arrangement as arrays. In order to test our hypothesis, we developed and applied an in-house Perl script that identified tRNA genes organization as an array unit. This survey included a total of 7,670 complete and drafts genomes of Mycobacterium genus, 4312 mycobacteriophage genomes and 40 mycobacteria plasmids. We showed that tRNA array units are abundant in genomes associated to the Mycobacterium genus, mainly in Mycobacterium abscessus complex species, being spread in chromosome, prophage, and plasmid genomes. Moreover, other non-coding RNA species (tmRNA and structured RNA) were also identified in these regions. Our results revealed that tRNA array units are not restrict, as previously assumed, to few bacteria phyla and genomes being present in one of the most diverse bacteria genus. We also provide a bioinformatics tool that allows further exploration of this issue in huge genomic databases. The presence of tRNA array units in plasmids and bacteriophages, associated with horizontal gene transfer, and in a bacteria genus that explores diverse niches, are indicatives that tRNA array units have impact in the bacteria biology.

Expression, purification and crystallization of phosphoribosyl transferase from a mycobacteriophage.

Tuberculosis (TB) continues to remain a leading cause of death globally. Of particular concern is the emergence and rise in incidence of multidrug-resistant and extremely drug-resistant cases of TB. To counter this threat, it is important to explore alternative therapies, including phage therapy. Phage BTCU-1 specifically infects Mycobacterium spp. and kills the majority of them. Intriguingly, many proteins from the phage do not share high amino-acid sequence identity with proteins from species other than phages. Here, the expression, purification and crystallization of one such protein, a putative phosphoribosyl transferase from phage BTCU-1, is reported. The crystals belonged to space group C2221, with unit-cell parameters a = 59.71, b = 64.42, c = 65.32 Å, α = ß = γ = 90°. The crystals diffracted X-rays to 2.2 Šresolution.

Isolation and characterization of bacteriophages from India, with lytic activity against Mycobacterium tuberculosis.

Bacteriophages are being considered as a promising natural resource for the development of alternative strategies against mycobacterial diseases, especially in the context of the wide-spread occurrence of drug resistance among the clinical isolates of Mycobacterium tuberculosis. However, there is not much information documented on mycobacteriophages from India. Here, we report the isolation of 17 mycobacteriophages using Mycobacterium smegmatis as the bacterial host, where 9 phages also lyse M. tuberculosis H37Rv. We present detailed analysis of one of these mycobacteriophages - PDRPv. Transmission electron microscopy and polymerase chain reaction analysis (of a conserved region within the TMP gene) show PDRPv to belong to the Siphoviridae family and B1 subcluster, respectively. The genome (69 110 bp) of PDRPv is circularly permuted double-stranded DNA with ∼66% GC content and has 106 open reading frames (ORFs). On the basis of sequence similarity and conserved domains, we have assigned function to 28 ORFs and have broadly categorized them into 6 groups that are related to replication and genome maintenance, DNA packaging, virion release, structural proteins, lysogeny-related genes and endolysins. The present study reports the occurrence of novel antimycobacterial phages in India and highlights their potential to contribute to our understanding of these phages and their gene products as potential antimicrobial agents.