Transcriptional Landscapes of Herelleviridae Bacteriophages and Staphylococcus aureus during Phage Infection: An Overview.

The issue of antibiotic resistance in healthcare worldwide has led to a pressing need to explore and develop alternative approaches to combat infectious diseases. Among these methods, phage therapy has emerged as a potential solution to tackle this growing challenge. Virulent phages of the Herelleviridae family, known for their ability to cause lysis of Staphylococcus aureus, a clinically significant pathogen frequently associated with multidrug resistance, have proven to be one of the most effective viruses utilized in phage therapy. In order to utilize phages for therapeutic purposes effectively, a thorough investigation into their physiology and mechanisms of action on infected cells is essential. The use of omics technologies, particularly total RNA sequencing, is a promising approach for analyzing the interaction between phages and their hosts, allowing for the assessment of both the behavior of the phage during infection and the cell's response. This review aims to provide a comprehensive overview of the physiology of the Herelleviridae family, utilizing existing analyses of their total phage transcriptomes. Additionally, it sheds light on the changes that occur in the metabolism of S. aureus when infected with virulent bacteriophages, contributing to a deeper understanding of the phage-host interaction.

A revised SNP-based barcoding scheme for typing Mycobacterium tuberculosis complex isolates.

The development of whole-genome sequencing technologies is gradually leading to a more detailed description of the population structure of the Mycobacterium tuberculosis complex (MTBC). In this study, we correlated previously published classifications on a collection of more than 10,000 genomes and proposed a new, comprehensive nomenclature that unifies the existing ones. In total, we identified 169 lineages and sublineages of M. tuberculosis/M. africanum and 9 animal-adapted species. For the purpose of organizing these genotypes in a more streamlined manner, we stratified them into five hierarchical levels. To represent the classification and compare it with the reference, we compiled a confirmatory data set of 670 high-quality isolates, which includes all genotypes and species of MTBC, and this confirmatory data set can serve as a basis for further studies. We proposed a set of 213 robust barcoding single-nucleotide polymorphisms and a suitable workflow for reliable differentiation of genotypes and species within the complex. This work integrates the results of all the major systematized studies to date to provide an understanding of the global diversity of the MTBC population structure. The results of this work may ultimately help to reliably determine the pathogen genotype and associate it with traits that reflect its prevalence, virulence, vaccination, and treatment efficiency, as well as to reliably find natural features revealed during its spread. IMPORTANCE Through years of research into the Mycobacterium tuberculosis complex (MTBC), a number of ambiguous phylogenetic classifications have emerged, which often overlap with one another. In the present study, we have combined all major studies on MTBC classification and inferred a unified, most complete to date classification and accompanying SNP barcodes.

Insight into pathogenomics and phylogeography of hypervirulent and highly-lethal Mycobacterium tuberculosis strain cluster.

BACKGROUND: . The Mycobacterium tuberculosis Beijing genotype is globally spread lineage with important medical properties that however vary among its subtypes. M. tuberculosis Beijing 14717-15-cluster was recently discovered as both multidrug-resistant, hypervirulent, and highly-lethal strain circulating in the Far Eastern region of Russia. Here, we aimed to analyze its pathogenomic features and phylogeographic pattern. RESULTS: . The study collection included M. tuberculosis DNA collected between 1996 and 2020 in different world regions. The bacterial DNA was subjected to genotyping and whole genome sequencing followed by bioinformatics and phylogenetic analysis. The PCR-based assay to detect specific SNPs of the Beijing 14717-15-cluster was developed and used for its screening in the global collections. Phylogenomic and phylogeographic analysis confirmed endemic prevalence of the Beijing 14717-15-cluster in the Asian part of Russia, and distant common ancestor with isolates from Korea (> 115 SNPs). The Beijing 14717-15-cluster isolates had two common resistance mutations RpsL Lys88Arg and KatG Ser315Thr and belonged to spoligotype SIT269. The Russian isolates of this cluster were from the Asian Russia while 4 isolates were from the Netherlands and Spain. The cluster-specific SNPs that significantly affect the protein function were identified in silico in genes within different categories (lipid metabolism, regulatory proteins, intermediary metabolism and respiration, PE/PPE, cell wall and cell processes). CONCLUSIONS: . We developed a simple method based on real-time PCR to detect clinically significant MDR and hypervirulent Beijing 14717-15-cluster. Most of the identified cluster-specific mutations were previously unreported and could potentially be associated with increased pathogenic properties of this hypervirulent M. tuberculosis strain. Further experimental study to assess the pathobiological role of these mutations is warranted.

PCR Assay for Rapid Taxonomic Differentiation of Virulent Staphylococcus aureus and Klebsiella pneumoniae Bacteriophages.

Phage therapy is now seen as a promising way to overcome the current global crisis in the spread of multidrug-resistant bacteria. However, phages are highly strain-specific, and in most cases one will have to isolate a new phage or search for a phage suitable for a therapeutic application in existing libraries. At an early stage of the isolation process, rapid screening techniques are needed to identify and type potential virulent phages. Here, we propose a simple PCR approach to differentiate between two families of virulent Staphylococcus phages (Herelleviridae and Rountreeviridae) and eleven genera of virulent Klebsiella phages (Przondovirus, Taipeivirus, Drulisvirus, Webervirus, Jiaodavirus, Sugarlandvirus, Slopekvirus, Jedunavirus, Marfavirus, Mydovirus and Yonseivirus). This assay includes a thorough search of a dataset comprising S. aureus (n = 269) and K. pneumoniae (n = 480) phage genomes available in the NCBI RefSeq/GenBank database for specific genes that are highly conserved at the taxonomic group level. The selected primers showed high sensitivity and specificity for both isolated DNA and crude phage lysates, which permits circumventing DNA purification protocols. Our approach can be extended and applied to any group of phages, given the large number of available genomes in the databases.

Isolation and Characterization of the First Zobellviridae Family Bacteriophage Infecting Klebsiella pneumoniae.

In order to address the upcoming crisis in the treatment of Klebsiella pneumoniae infections, caused by an increasing proportion of resistant isolates, new approaches to antimicrobial therapy must be developed. One approach would be to use (bacterio)phages and/or phage derivatives for therapy. In this study, we present a description of the first K. pneumoniae phage from the Zobellviridae family. The vB_KpnP_Klyazma podovirus, which forms translucent halos around the plaques, was isolated from river water. The phage genome is composed of 82 open reading frames, which are divided into two clusters located on opposite strands. Phylogenetic analysis revealed that the phage belongs to the Zobellviridae family, although its identity with the closest member of this family was not higher than 5%. The bacteriophage demonstrated lytic activity against all (n = 11) K. pneumoniae strains with the KL20 capsule type, but only the host strain was lysed effectively. The receptor-binding protein of the phage was identified as a polysaccharide depolymerase with a pectate lyase domain. The recombinant depolymerase protein showed concentration-dependent activity against all strains with the KL20 capsule type. The ability of a recombinant depolymerase to cleave bacterial capsular polysaccharides regardless of a phage's ability to successfully infect a particular strain holds promise for the possibility of using depolymerases in antimicrobial therapy, even though they only make bacteria sensitive to environmental factors, rather than killing them directly.

The Gene Expression Profile Differs in Growth Phases of the Bifidobacterium Longum Culture.

To date, transcriptomics have been widely and successfully employed to study gene expression in different cell growth phases of bacteria. Since bifidobacteria represent a major component of the gut microbiota of a healthy human that is associated with numerous health benefits for the host, it is important to study them using transcriptomics. In this study, we applied the RNA-Seq technique to study global gene expression of B. longum at different growth phases in order to better understand the response of bifidobacterial cells to the specific conditions of the human gut. We have shown that in the lag phase, ABC transporters, whose function may be linked to active substrate utilization, are increasingly expressed due to preparation for cell division. In the exponential phase, the functions of activated genes include synthesis of amino acids (alanine and arginine), energy metabolism (glycolysis/gluconeogenesis and nitrogen metabolism), and translation, all of which promote active cell division, leading to exponential growth of the culture. In the stationary phase, we observed a decrease in the expression of genes involved in the control of the rate of cell division and an increase in the expression of genes involved in defense-related metabolic pathways. We surmise that the latter ensures cell survival in the nutrient-deprived conditions of the stationary growth phase.

Genome-Wide Transcriptional Response of Mycobacterium smegmatis MC2155 to G-Quadruplex Ligands BRACO-19 and TMPyP4.

G-quadruplexes (G4s) are non-canonical DNA structures that could be considered as potential therapeutic targets for antimicrobial compounds, also known as G4-stabilizing ligands. While some of these ligands are shown in vitro to have a stabilizing effect, the precise mechanism of antibacterial action has not been fully investigated. Here, we employed genome-wide RNA-sequencing to analyze the response of Mycobacterium smegmatis to inhibitory concentrations of BRACO-19 and TMPyP4 G4 ligands. The expression profile changed (FDR < 0.05, log2FC > |1|) for 822 (515↑; 307↓) genes in M. smegmatis in response to BRACO-19 and for 680 (339↑; 341↓) genes in response to TMPyP4. However, the analysis revealed no significant ligand-induced changes in the expression levels of G4-harboring genes, genes under G4-harboring promoters, or intergenic regions located on mRNA-like or template strands. Meanwhile, for the BRACO-19 ligand, we found significant changes in the replication and repair system genes, as well as in iron metabolism genes which is, undoubtedly, evidence of the induced stress. For the TMPyP4 compound, substantial changes were found in transcription factors and the arginine biosynthesis system, which may indicate multiple biological targets for this compound.

Global Transcriptomic Response of Staphylococcus aureus to Virulent Bacteriophage Infection.

In light of the ever-increasing number of multidrug-resistant bacteria worldwide, bacteriophages are becoming a valid alternative to antibiotics; therefore, their interactions with host bacteria must be thoroughly investigated. Here, we report genome-wide transcriptional changes in a clinical Staphylococcus aureus SA515 strain for three time points after infection with the vB_SauM-515A1 kayvirus. Using an RNA sequencing approach, we identify 263 genes that were differentially expressed (DEGs) between phage-infected and uninfected host samples. Most of the DEGs were identified at an early stage of phage infection and were mainly involved in nucleotide and amino acid metabolism, as well as in cell death prevention. At the subsequent infection stages, the vast majority of DEGs were upregulated. Interestingly, 39 upregulated DEGs were common between the 15th and 30th minutes post-infection, and a substantial number of them belonged to the prophages. Furthermore, some virulence factors were overexpressed at the late infection stage, which necessitates more stringent host strain selection requirements for further use of bacteriophages for therapeutic purposes. Thus, this work allows us to better understand the influence of kayviruses on the metabolic systems of S. aureus and contributes to a better comprehension of phage therapy.

Substitutions in SurA and BamA Lead to Reduced Susceptibility to Broad Range Antibiotics in Gonococci.

There is growing concern about the emergence and spread of multidrug-resistant Neisseria gonorrhoeae. To effectively control antibiotic-resistant bacterial pathogens, it is necessary to develop new antimicrobials and to understand the resistance mechanisms to existing antibiotics. In this study, we discovered the unexpected onset of drug resistance in N. gonorrhoeae caused by amino acid substitutions in the periplasmic chaperone SurA and the ß-barrel assembly machinery component BamA. Here, we investigated the i19.05 clinical isolate with mutations in corresponding genes along with reduced susceptibility to penicillin, tetracycline, and azithromycin. The mutant strain NG05 (surAmut bamAmut, and penAmut) was obtained using the pan-susceptible n01.08 clinical isolate as a recipient in the transformation procedure. Comparative proteomic analysis of NG05 and n01.08 strains revealed significantly increased levels of other chaperones, Skp and FkpA, and some transport proteins. Efflux pump inhibition experiments demonstrated that the reduction in sensitivity was achieved due to the activity of efflux pumps. We hypothesize that the described mutations in the surA and bamA genes cause the qualitative and quantitative changes of periplasmic chaperones, which in turn alters the function of synthesized cell envelope proteins.

Novel Klebsiella pneumoniae K23-Specific Bacteriophages From Different Families: Similarity of Depolymerases and Their Therapeutic Potential.

Antibiotic resistance is a major public health concern in many countries worldwide. The rapid spread of multidrug-resistant (MDR) bacteria is the main driving force for the development of novel non-antibiotic antimicrobials as a therapeutic alternative. Here, we isolated and characterized three virulent bacteriophages that specifically infect and lyse MDR Klebsiella pneumoniae with K23 capsule type. The phages belonged to the Autographiviridae (vB_KpnP_Dlv622) and Myoviridae (vB_KpnM_Seu621, KpS8) families and contained highly similar receptor-binding proteins (RBPs) with polysaccharide depolymerase enzymatic activity. Based on phylogenetic analysis, a similar pattern was also noted for five other groups of depolymerases, specific against capsule types K1, K30/K69, K57, K63, and KN2. The resulting recombinant depolymerases Dep622 (phage vB_KpnP_Dlv622) and DepS8 (phage KpS8) demonstrated narrow specificity against K. pneumoniae with capsule type K23 and were able to protect Galleria mellonella larvae in a model infection with a K. pneumoniae multidrug-resistant strain. These findings expand our knowledge of the diversity of phage depolymerases and provide further evidence that bacteriophages and phage polysaccharide depolymerases represent a promising tool for antimicrobial therapy.

Transcriptomic Profile of Mycobacterium smegmatis in Response to an Imidazo[1,2-b][1,2,4,5]tetrazine Reveals Its Possible Impact on Iron Metabolism.

Tuberculosis (TB), caused by the Mycobacterium tuberculosis complex bacteria, is one of the most pressing health problems. The development of new drugs and new therapeutic regimens effective against the pathogen is one of the greatest challenges in the way of tuberculosis control. Imidazo[1,2-b][1,2,4,5]tetrazines have shown promising activity against M. tuberculosis and M. smegmatis strains. Mutations in MSMEG_1380 lead to mmpS5-mmpL5 operon overexpression, which provides M. smegmatis with efflux-mediated resistance to imidazo[1,2-b][1,2,4,5]tetrazines, but the exact mechanism of action of these compounds remains unknown. To assess the mode of action of imidazo[1,2-b][1,2,4,5]tetrazines, we analyzed the transcriptomic response of M. smegmatis to three different concentrations of 3a compound: 1/8×, 1/4×, and 1/2× MIC. Six groups of genes responsible for siderophore synthesis and transport were upregulated in a dose-dependent manner, while virtual docking revealed proteins involved in siderophore synthesis as possible targets for 3a. Thus, we suggest that imidazo[1,2-b][1,2,4,5]tetrazines may affect mycobacterial iron metabolism.

MDR and Pre-XDR Clinical Mycobacterium tuberculosis Beijing Strains: Assessment of Virulence and Host Cytokine Response in Mice Infectious Model.

Mycobacterium tuberculosis Beijing genotype associated with drug resistance is a growing public health problem worldwide. The aim of this study was the assessment of virulence for C57BL/6 mice after infection by clinical M. tuberculosis strains 267/47 and 120/26, which belong to the modern sublineages B0/W148 and Central Asia outbreak of the Beijing genotype, respectively. The sublineages were identified by the analysis of the strains' whole-genomes. The strains 267/47 and 120/26 were characterized as agents of pre-extensively drug-resistant (pre-XDR) and multidrug-resistant (MDR) tuberculosis, respectively. Both clinical strains were slow-growing in 7H9 broth compared to the M. tuberculosis H37Rv strain. The survival rates of C57BL/6 mice infected by 267/47, 120/26, and H37Rv on the 150th day postinfection were 10%, 40%, and 70%, respectively. Mycobacterial load in the lungs, spleen, and liver was higher and histopathological changes were more expressed for mice infected by the 267/47 strain compared to those infected by the 120/26 and H37Rv strains. The cytokine response in the lungs of C57BL/6 mice after infection with the 267/47, 120/26, and H37Rv strains was different. Notably, proinflammatory cytokine genes Il-1α, Il-6, Il-7, and Il-17, as well as anti-inflammatory genes Il-6 and Il-13, were downregulated after an infection caused by the 267/47 strain compared to those after infection with the H37Rv strain.

Gene Networks Underlying the Resistance of Bifidobacterium longum to Inflammatory Factors.

As permanent residents of the normal gut microbiota, bifidobacteria have evolved to adapt to the host's immune response whose priority is to eliminate pathogenic agents. The mechanisms that ensure the survival of commensals during inflammation and maintain the stability of the core component of the normal gut microbiota in such conditions remain poorly understood. We propose a new in vitro approach to study the mechanisms of resistance to immune response factors based on high-throughput sequencing followed by transcriptome analysis. This approach allowed us to detect differentially expressed genes associated with inflammation. In this study, we demonstrated that the presence of the pro-inflammatory cytokines IL-6 and TNFα to the growth medium of the B. longum subsp. longum GT15 strain changes the latter's growth rate insignificantly while affecting the expression of certain genes. We identified these genes and performed a COG and a KEGG pathway enrichment analysis. Using phylogenetic profiling we predicted the operons of genes whose expression was triggered by the cytokines TNFα and IL-6 in vitro. By mapping the transcription start points, we experimentally validated the predicted operons. Thus, in this study, we predicted the genes involved in a putative signaling pathway underlying the mechanisms of resistance to inflammatory factors in bifidobacteria. Since bifidobacteria are a major component of the human intestinal microbiota exhibiting pronounced anti-inflammatory properties, this study is of great practical and scientific relevance.

Transcriptional Landscape of Staphylococcus aureus Kayvirus Bacteriophage vB_SauM-515A1.

The Twort-like myoviruses (Kayvirus genus) of S. aureus are promising agents for bacteriophage therapy due to a broad host range and high killing activity against clinical isolates. This work improves the current understanding of the phage infection physiology by transcriptome analysis. The expression profiles of a typical member of the Kayvirus genus (vB_SauM-515A1) were obtained at three time-points post-infection using RNA sequencing. A total of 35 transcription units comprising 238 ORFs were established. The sequences for 58 early and 12 late promoters were identified in the phage genome. The early promoters represent the strong sigma-70 promoters consensus sequence and control the host-dependent expression of 26 transcription units (81% of genes). The late promoters exclusively controlled the expression of four transcription units, while the transcription of the other five units was directed by both types of promoters. The characteristic features of late promoters were long -10 box of TGTTATATTA consensus sequence and the absence of -35 boxes. The data obtained are also of general interest, demonstrating a strategy of the phage genome expression with a broad overlap of the early and late transcription phases without any middle transcription, which is unusual for the large phage genomes (>100 kbp).

Contribution of Podoviridae and Myoviridae bacteriophages to the effectiveness of anti-staphylococcal therapeutic cocktails.

Bacteriophage therapy is considered one of the most promising therapeutic approaches against multi-drug resistant bacterial infections. Infections caused by Staphylococcus aureus are very efficiently controlled with therapeutic bacteriophage cocktails, containing a number of individual phages infecting a majority of known pathogenic S. aureus strains. We assessed the contribution of individual bacteriophages comprising a therapeutic bacteriophage cocktail against S. aureus in order to optimize its composition. Two lytic bacteriophages vB_SauM-515A1 (Myoviridae) and vB_SauP-436A (Podoviridae) were isolated from the commercial therapeutic cocktail produced by Microgen (Russia). Host ranges of the phages were established on the panel of 75 S. aureus strains. Phage vB_SauM-515A1 lysed 85.3% and vB_SauP-436A lysed 68.0% of the strains, however, vB_SauP-436A was active against four strains resistant to vB_SauM-515A1, as well as to the therapeutic cocktail per se. Suboptimal results of the therapeutic cocktail application were due to extremely low vB_SauP-436A1 content in this composition. Optimization of the phage titers led to an increase in overall cocktail efficiency. Thus, one of the effective ways to optimize the phage cocktails design was demonstrated and realized by using bacteriophages of different families and lytic spectra.

Aureolic Acid Group of Agents as Potential Antituberculosis Drugs.

Mycobacterium tuberculosis is one of the most dangerous pathogens. Bacterial resistance to antituberculosis drugs grows each year, but searching for new drugs is a long process. Testing for available drugs to find active against mycobacteria may be a good alternative. In this work, antibiotics of the aureolic acid group were tested on a model organism Mycobacterium smegmatis. We presumed that antibiotics of this group may be potential G4 ligands. However, this was not confirmed in our analyses. We determined the antimicrobial activity of these drugs and revealed morphological changes in the cell structure upon treatment. Transcriptomic analysis documented increased expression of MSMEG_3743/soj and MSMEG_4228/ftsW, involved in cell division. Therefore, drugs may affect cell division, possibly disrupting the function of the Z-ring and the formation of a septum. Additionally, a decrease in the transcription level of several indispensable genes, such as nitrate reductase subunits (MSMEG_5137/narI and MSMEG_5139/narX) and MSMEG_3205/hisD was shown. We concluded that the mechanism of action of aureolic acid and its related compounds may be similar to that bedaquiline and disturb the NAD+/NADH balance in the cell. All of this allowed us to conclude that aureolic acid derivatives can be considered as potential antituberculosis drugs.

Author Correction: System OMICs analysis of Mycobacterium tuberculosis Beijing B0/W148 cluster.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Metabolic Changes of Mycobacterium tuberculosis during the Anti-Tuberculosis Therapy.

Tuberculosis, caused by Mycobacterium tuberculosis complex bacteria, remains one of the most pressing health problems. Despite the general trend towards reduction of the disease incidence rate, the situation remains extremely tense due to the distribution of the resistant forms. Most often, these strains emerge through the intra-host microevolution of the pathogen during treatment failure. In the present study, the focus was on three serial clinical isolates of Mycobacterium tuberculosis Beijing B0/W148 cluster from one patient with pulmonary tuberculosis, to evaluate their changes in metabolism during anti-tuberculosis therapy. Using whole genome sequencing (WGS), 9 polymorphisms were determined, which occurred in a stepwise or transient manner during treatment and were linked to the resistance (GyrA D94A; inhA t-8a) or virulence. The effect of the inhA t-8a mutation was confirmed on both proteomic and transcriptomic levels. Additionally, the amount of RpsL protein, which is a target of anti-tuberculosis drugs, was reduced. At the systemic level, profound changes in metabolism, linked to the evolution of the pathogen in the host and the effects of therapy, were documented. An overabundance of the FAS-II system proteins (HtdX, HtdY) and expression changes in the virulence factors have been observed at the RNA and protein levels.

System OMICs analysis of Mycobacterium tuberculosis Beijing B0/W148 cluster.

Mycobacterium tuberculosis Beijing B0/W148 is one of the most widely distributed clusters in the Russian Federation and in some countries of the former Soviet Union. Recent studies have improved our understanding of the reasons for the "success" of the cluster but this area remains incompletely studied. Here, we focused on the system omics analysis of the RUS_B0 strain belonging to the Beijing B0/W148 cluster. Completed genome sequence of RUS_B0 (CP020093.1) and a collection of WGS for 394 cluster strains were used to describe the main genetic features of the population. In turn, proteome and transcriptome studies allowed to confirm the genomic data and to identify a number of finds that have not previously been described. Our results demonstrated that expression of the whiB6 which contains cluster-specific polymorphism (a151c) increased almost 40 times in RUS_B0. Additionally, the level of ethA transcripts in RUS_B0 was increased by more than 7 times compared to the H37Rv. Start sites for 10 genes were corrected based on the combination of proteomic and transcriptomic data. Additionally, based on the omics approach, we identified 5 new genes. In summary, our analysis allowed us to summarize the available results and also to obtain fundamentally new data.