Genomic analysis of the international high-risk clonal lineage Klebsiella pneumoniae sequence type 395.

BACKGROUND: Klebsiella pneumoniae, which is frequently associated with hospital- and community-acquired infections, contains multidrug-resistant (MDR), hypervirulent (hv), non-MDR/non-hv as well as convergent representatives. It is known that mostly international high-risk clonal lineages including sequence types (ST) 11, 147, 258, and 307 drive their global spread. ST395, which was first reported in the context of a carbapenemase-associated outbreak in France in 2010, is a less well-characterized, yet emerging clonal lineage. METHODS: We computationally analyzed a large collection of K. pneumoniae ST395 genomes (n = 297) both sequenced in this study and reported previously. By applying multiple bioinformatics tools, we investigated the core-genome phylogeny and evolution of ST395 as well as distribution of accessory genome elements associated with antibiotic resistance and virulence features. RESULTS: Clustering of the core-SNP alignment revealed four major clades with eight smaller subclades. The subclades likely evolved through large chromosomal recombination, which involved different K. pneumoniae donors and affected, inter alia, capsule and lipopolysaccharide antigen biosynthesis regions. Most genomes contained acquired resistance genes to extended-spectrum cephalosporins, carbapenems, and other antibiotic classes carried by multiple plasmid types, and many were positive for hypervirulence markers, including the siderophore aerobactin. The detection of "hybrid" resistance and virulence plasmids suggests the occurrence of the convergent ST395 pathotype. CONCLUSIONS: To the best of our knowledge, this is the first study that investigated a large international collection of K. pneumoniae ST395 genomes and elucidated phylogenetics and detailed genomic characteristics of this emerging high-risk clonal lineage.

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.

The Origin of Genetic Code and Translation in the Framework of Current Concepts on the Origin of Life.

The origin of genetic code and translation system is probably the central and most difficult problem in the investigations on the origin of life and one of the most complex problems in the evolutionary biology in general. There are multiple hypotheses on the emergence and development of existing genetic systems that propose the mechanisms for the origin and early evolution of genetic code, as well as for the emergence of replication and translation. Here, we discuss the most well-known of these hypotheses, although none of them provides a description of the early evolution of genetic systems without gaps and assumptions. The RNA world hypothesis is a currently prevailing scientific idea on the early evolution of biological and pre-biological structures, the main advantage of which is the assumption that RNAs as the first living systems were self-sufficient, i.e., capable of functioning as both catalysts and templates. However, this hypothesis has also significant limitations. In particular, no ribozymes with processive polymerase activity have been yet discovered or synthesized. Taking into account the mutual need of proteins and nucleic acids in each other in the current world, many authors propose the early evolution scenarios based on the co-evolution of these two classes of organic molecules. They postulate that the emergence of translation was necessary for the replication of nucleic acids, in contrast to the RNA world hypothesis, according to which the emergence of translation was preceded by the era of self-replicating RNAs. Although such scenarios are less parsimonious from the evolutionary point of view, since they require simultaneous emergence and evolution of two classes of organic molecules, as well as the emergence of synchronized replication and translation, their major advantage is that they explain the development of processive and much more accurate protein-dependent replication.

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.

FN3 protein fragment containing two type III fibronectin domains from B. longum GT15 binds to human tumor necrosis factor alpha in vitro.

Most species of the genus Bifidobacterium contain the gene cluster PFNA, which is presumably involved in the species-specific communication between bacteria and their hosts. The gene cluster PFNA consists of five genes including fn3, which codes for a protein containing two fibronectin type III domains. Each fibronectin domain contains sites similar to cytokine-binding sites of human receptors. Based on this finding we assumed that this protein would bind specifically to human cytokines in vitro. We cloned a fragment of the fn3 gene (1503 bp; 501 aa) containing two fibronectin domains, from the strain B. longum subsp. longum GT15. After cloning the fragment into the expression vector pET16b and expressing it in E. coli, the protein product was purified to a homogenous state for further analysis. Using the immunoferment method, we tested the purified fragment's ability to bind the following human cytokines: IL-1ß, IL-6, IL-10, TNFα. We developed a sandwich ELISA system to detect any specific interactions between the purified protein and any of the studied cytokines. We found that the purified protein fragment only binds to TNFα.

Positive Selection in Bifidobacterium Genes Drives Species-Specific Host-Bacteria Communication.

Bifidobacteria are commensal microorganisms that inhabit a wide range of hosts, including insects, birds and mammals. The mechanisms responsible for the adaptation of bifidobacteria to various hosts during the evolutionary process remain poorly understood. Previously, we reported that the species-specific PFNA gene cluster is present in the genomes of various species of the Bifidobacterium genus. The cluster contains signal transduction and adhesion genes that are presumably involved in the communication between bifidobacteria and their hosts. The genes in the PFNA cluster show high sequence divergence between bifidobacterial species, which may be indicative of rapid evolution that drives species-specific adaptation to the host organism. We used the maximum likelihood approach to detect positive selection in the PFNA genes. We tested for both pervasive and episodic positive selection to identify codons that experienced adaptive evolution in all and individual branches of the Bifidobacterium phylogenetic tree, respectively. Our results provide evidence that episodic positive selection has played an important role in the divergence process and molecular evolution of sequences of the species-specific PFNA genes in most bifidobacterial species. Moreover, we found the signatures of pervasive positive selection in the molecular evolution of the tgm gene in all branches of the Bifidobacterium phylogenetic tree. These results are consistent with the suggested role of PFNA gene cluster in the process of specific adaptation of bifidobacterial species to various hosts.

Draft Genome Sequences of Bifidobacterium angulatum GT102 and Bifidobacterium adolescentis 150: Focusing on the Genes Potentially Involved in the Gut-Brain Axis.

The draft genome sequences of Bifidobacterium angulatum GT102 and Bifidobacterium adolescentis 150 strains isolated from the human intestinal microbiota are reported. Both strains are able to produce gamma-aminobutyric acid (GABA). Detailed genomes analysis will help to understand the role of GABA in the functioning of gut-brain axis.