Nomeda Kuisiene

Actinobacteria of the genus Frigoribacterium were isolated from adelgid Adelges (Aphrastasia) pectinatae collected from a Korean fir tree. Genomic analyses revealed that these bacteria encode a range of factors that may be involved in the interactions among Frigoribacterium strains, adelgids and/or conifers. Secreted carbohydrate‐active enzymes were identified in the genomes, which allow these bacteria to degrade plant polysaccharides such as cellulose, xylan, pectin and mannan, the main hemicellulose component of softwood. The degradation potential of insect cuticles was investigated, and secreted chitinases belonging to the GH18 family were predicted to be present in the genomes. However, no phenotypic chitinolytic activity was detected. The potential interactions between these bacterial strains and either plants or insects were assessed, resulting in a few high‐scoring hits. The related Frigoribacterium genomes were compared, revealing several unique features, such as numerous orthologous gene clusters specific to these strains and five biosynthetic gene clusters. Ten genomic islands were predicted in the genomes of the adelgid‐associated strains, which contained genes responsible for adapting to environmental changes, resisting heavy metals and expanding metabolic capabilities. We propose a new species, Frigoribacterium adelgis , belonging to the genus Frigoribacterium , based on these results.

Listeria monocytogenes is a saprophytic gram-positive bacterium and opportunistic foodborne pathogen that can cause listeriosis in humans. The incidence of listeriosis has been rising globally and, despite antimicrobial treatment, the mortality rates associated with the most severe forms of listeriosis such as sepsis, meningitis and meningoencephalitis remain high. The notification of listeriosis in humans is mandatory in Lithuania, and up to 20 cases are reported annually. However, no studies have described the detailed virulence and antimicrobial susceptibility profiles of any clinical L. monocytogenes strains in Lithuania. Accordingly, this study aimed to describe the antibiotic susceptibility of invasive L. monocytogenes and perform in-depth characterization of strains isolated from patients with neuroinfections through whole-genome sequencing. A total of 70 isolates were collected, mostly from infected patients aged 65 or older, between 2016 and 2021 : 41 (58.6%) from blood, 19 (27.1%) from cerebrospinal fluid, 5 (7.1%) from wounds, 1 (1.4%) from pleural fluid and 1 (1.4%) from a brain abscess. Two phylogenetic lineages were identified—I (n = 16/70, 22.9%) and II (n = 54/70, 77.1%)—along with three serogroups—IIa (n = 53/70, 75.7%), IVb (n = 16/70, 22.9%), and IIc (n = 1/70, 1.4%). Genomic analysis of 20 isolates showed a high level of diversity with seven genotypes: ST6 (n = 6), ST155 (n = 5), ST8 (n = 4), ST504 (n = 2) and singletons for ST37, ST451 and ST2. Phylogenetic analysis clustered these isolates into two clades defined by serogroups IVb and IIa. Notably, five isolates were clustered tightly together (difference of 6–48 core SNPs from reference and 0, 4 or 44 SNPs from each other) with ST155, previously reported in a European outbreak. Comparison with publicly available L. monocytogenes genomes did not identify unique clusters or genotypes. No acquired antimicrobial resistance genes were identified. Our study highlights the complementary value of whole-genome sequencing in routine PCR-based surveillance in Lithuania. This is the first study to characterize and compare genomes for L. monocytogenes associated with neuroinfections in Lithuania using whole-genome sequencing. The retrospective detection of the ST155 clone underscores the need for a review and strengthening of epidemiological surveillance strategies in clinical and non-clinical settings in Lithuania.

A high temperature-adapted bacteriophage, vB_PtoS_NIIg3.2 (NIIg3.2), was isolated in Lithuania from compost heaps using Parageobacillus toebii strain NIIg-3 as a host for phage propagation. Furthermore, NIIg3.2 was active against four strains of Geobacillus thermodenitrificans, and it infected the host cells from 50 to 80 °C. Transmission electron microscopy analysis revealed siphovirus morphology characterized by an isometric head (~59 nm in diameter) and a noncontractile tail (~226 nm in length). The double-stranded DNA genome of NIIg3.2 (38,970 bp) contained 71 probable protein-encoding genes and no genes for tRNA. In total, 29 NIIg3.2 ORFs were given a putative functional annotation, including those coding for the proteins responsible for DNA packaging, virion structure/morphogenesis, phage–host interactions, lysis/lysogeny, replication/regulation, and nucleotide metabolism. Based on comparative phylogenetic and bioinformatic analysis, NIIg3.2 cannot be assigned to any genus currently recognized by ICTV and potentially represents a new one within siphoviruses. The results of this study not only extend our knowledge about poorly explored thermophilic bacteriophages but also provide new insights for further investigation and understanding the evolution of Bacilllus-group bacteria-infecting viruses.

We report a detailed characterization of five thermophilic bacteriophages (phages) that were isolated from compost heaps in Vilnius, Lithuania using Geobacillus thermodenitrificans strains as the hosts for phage propagation. The efficiency of plating experiments revealed that phages formed plaques from 45 to 80 °C. Furthermore, most of the phages formed plaques surrounded by halo zones, indicating the presence of phage-encoded bacterial exopolysaccharide (EPS)-degrading depolymerases. Transmission Electron Microscopy (TEM) analysis revealed that all phages were siphoviruses characterized by an isometric head (from ~63 nm to ~67 nm in diameter) and a non-contractile flexible tail (from ~137 nm to ~150 nm in length). The genome sequencing resulted in genomes ranging from 38,161 to 39,016 bp. Comparative genomic and phylogenetic analysis revealed that all the isolated phages had no close relatives to date, and potentially represent three new genera within siphoviruses. The results of this study not only improve our knowledge about poorly explored thermophilic bacteriophages but also give new insights for further investigation of thermophilic and/or thermostable enzymes of bacterial viruses.

Extraintestinal pathogenic Escherichia coli (ExPEC) isolates are a main cause of bloodstream infections. The aim of this study was to characterize 256 β-lactam–resistant, bacteremia-causing E. coli isolates collected from 12 healthcare institutions in Lithuania in 2014 and 2018. All isolates were identified as E. coli via MALDI-TOF MS and VITEK ^®2. In addition, the isolates were analyzed for the presence of 29 resistance genes and 13 virulence genes, divided into phylogenetic groups (A, B1, B2, C, D, E, and F), and characterized using rep-PCR genotyping methods (BOX-PCR and (GTG)₅-PCR). Analyzing the results of this study showed tetA-strB-sul2-TEM-NDM-strA-fosA-AIM-sul3-aadA-CTX-M-9 to be the most common resistance gene combination (67.2% of all isolates). Additionally, the most common virulence genes established were fimH (98.4% of all isolates), fyuA (91.8%), and traT (81.3%) and the most common gene combination was fuyA-fimH-iroN (58.6% of all isolates). Next, the isolates were separated into four phylogenetic groups: A, B1, B2, and F, where group A isolates were detected at a significantly higher frequency (79.3% of all isolates). Finally, a total of 235 genotyping profiles were established using rep-PCR methods, and all profiles were separated into fourteen genotypic clusters, with each cluster containing profiles with a variety of virulence and resistance genes not restricted to any specific cluster. The results of this study elucidate E. coli antimicrobial resistance patterns by highlighting the variability and diversity of resistance and virulence genes and providing phylogenetic classification, genetic profiling, and clustering data. These results may improve clinical control of multidrug-resistant infections in healthcare institutions and contribute to the prevention of potential outbreaks.

Bacterial resistance to antimicrobial agents plays an important role in the treatment of bacterial infections in healthcare institutions. The spread of multidrug-resistant bacteria can occur during inter- and intra-hospital transmissions among patients and hospital personnel. For this reason, more studies must be conducted to understand how resistance occurs in bacteria and how it moves between hospitals by comparing data from different years and looking out for any patterns that might emerge. Multidrug-resistant (MDR) Acinetobacter spp. was studied at 14 healthcare institutions in Lithuania during 2014, 2016, and 2018 using samples from human bloodstream infections. In total, 194 isolates were collected and identified using MALDI-TOF and VITEK2 analyzers as Acinetobacter baumannii group bacteria. After that, the isolates were analyzed for the presence of different resistance genes (20 genes were analyzed) and characterized by using the Rep-PCR and MLVA (multiple-locus variable-number tandem repeat analysis) genotyping methods. The results of the study showed the relatedness of the different Acinetobacter spp. isolates and a possible circulation of resistance genes or profiles during the different years of the study. This study provides essential information, such as variability and diversity of resistance genes, genetic profiling, and clustering of isolates, to better understand the antimicrobial resistance patterns of Acinetobacter spp. These results can be used to strengthen the control of multidrug-resistant infections in healthcare institutions and to prevent potential outbreaks of this pathogen in the future.

The genome sequencing and mining of microorganisms from unexplored and extreme environments has become important in the process of identifying novel biosynthetic pathways. In the present study, the biosynthetic potential of Paenibacillus sp. strains 23TSA30-6 and 28ISP30-2 was investigated. Both strains were isolated from the deep oligotrophic Krubera-Voronja Cave and were found to be highly active against both Gram-positive and Gram-negative bacteria. Genome mining revealed a high number of biosynthetic gene clusters in the cave strains: 21 for strain 23TSA30-6 and 19 for strain 28ISP30-2. Single clusters encoding the biosynthesis of phosphonate, terpene, and siderophore, as well as a single trans-AT polyketide synthase/non-ribosomal peptide synthetase, were identified in both genomes. The most numerous clusters were assigned to the biosynthetic pathways of non-ribosomal peptides and ribosomally synthesized and post-translationally modified peptides. Although four non-ribosomal peptide synthetase gene clusters were predicted to be involved in the biosynthesis of known compounds (fusaricidin, polymyxin B, colistin A, and tridecaptin) of the genus Paenibacillus, discrepancies in the structural organization of the clusters, as well as in the substrate specificity of some adenylation domains, were detected between the reference pathways and the clusters in our study. Among the clusters involved in the biosynthesis of ribosomally synthesized peptides, only one was predicted to be involved in the biosynthesis of a known compound: paenicidin B. Most biosynthetic gene clusters in the genomes of the cave strains showed a low similarity with the reference pathways and were predicted to represent novel biosynthetic pathways. In addition, the cave strains differed in their potential to encode the biosynthesis of a few unique, previously unknown compounds (class II lanthipeptides and three non-ribosomal peptides). The phenotypic characterization of proteinaceous and volatile compounds produced by strains 23TSA30-6 and 28ISP30-2 was also performed, and the results were compared with those of genome mining.

Caves are considered to be extreme and challenging environments. It is believed that the ability of microorganisms to produce secondary metabolites enhances their survivability and adaptiveness in the energy-starved cave environment. Unfortunately, information on the genetic potential for the production of secondary metabolites, such as polyketides and nonribosomal peptides, is limited. In the present study, we aimed to identify and characterize genes responsible for the production of secondary metabolites in the microbial community of one of the deepest caves in the world, Krubera-Voronja Cave (43.4184 N 40.3083 E, Western Caucasus). The analysed sample materials included sediments, drinkable water from underground camps, soil and clay from the cave walls, speleothems and coloured spots from the cave walls. The type II polyketide synthases (PKSs) ketosynthases α and β and the adenylation domains of nonribosomal peptide synthetases (NRPSs) were investigated using a metagenomic approach. Taxonomic diversity analysis showed that most PKS sequences could be attributed to Actinobacteria followed by unclassified bacteria and Acidobacteria, while the NRPS sequences were more taxonomically diverse and could be assigned to Proteobacteria, Actinobacteria, Cyanobacteria, Firmicutes, Chloroflexi, etc. Only three putative metabolites could be predicted: an angucycline group polyketide, a massetolide A-like cyclic lipopeptide and a surfactin-like lipopeptide. The absolute majority of PKS and NRPS sequences showed low similarity with the sequences of the reference biosynthetic pathways, suggesting that these sequences could be involved in the production of novel secondary metabolites.

Identification of novel bioactive compounds represents an important field in modern biomedical research. Microorganisms of the underexplored environments, such as deserts, hot springs, oceans, and caves are highly promising candidates for screening such metabolites. Screening for biosynthetic genes is the most effective strategy to characterize bioactivity in a certain environment. However, knowledge is either scant or non-existent about the expression of the biosynthetic genes encoding for various bioactive compounds in the microorganisms from the caves. The aim of the current study was to screen for the genes of polyketide synthases and non-ribosomal peptide synthetases in Krubera–Voronja Cave (43.4184 N 40.3083 E, Western Caucasus) bacterial isolates as well as to evaluate the expression of these genes under laboratory conditions. In total, 91 bacterial strains isolated from the cave were screened for the presence of polyketide synthase and non-ribosomal peptide synthetase genes. Phenotypically inactive strains were the main focus (the test group) of our study, while the strains with the identified antibacterial activity served as the control group. Our PCR-based screening clearly showed that the majority of the strains harbored at least one biosynthetic gene. Prediction of the putative products allowed us to identify bioactive compounds with antibacterial, anticancer, antifungal, anti-inflammatory, antimycoplasmic, antiviral, insecticidal, and thrombolytic activity. For most polyketide synthases and non-ribosomal peptide synthetases, putative products could not be predicted; they are unknown. Qualitative transcriptional analysis did not show substantial differences between the test group and the control group of the strains. One to four biosynthetic genes were constitutively expressed in all the tested strains, irrespective of the group. Quantitative transcriptional analysis of the constitutively expressed biosynthetic genes demonstrated that the expression of a particular gene could be affected by both the amount of the nutrients in the culture medium and the growth phase.

Clostridioides difficile has become the leading nosocomial Gram-positive pathogen in the developed countries. In Lithuania, the national surveillance program for C. difficile started in 2017. Enzyme immunoassay, the real-time PCR system, and culture are used for laboratory confirmation of C. difficile infection in Lithuanian clinical laboratories. No reference laboratory for C. difficile is present in Lithuania. Fifty-eight isolates of C. difficile were collected in 2016 and 2017 in two hospitals using real-time PCR and culture methods. Agarose gel–based PCR ribotyping, multilocus variable number tandem repeats analysis (MLVA), and multilocus sequence typing (MLST) were used for the genotypic characterization of 28 isolates. PCR ribotyping and MLST showed that 78.6% of the tested toxigenic isolates belong to the ribotype RT027/ST1. Using MLVA, 95.5% of RT027 isolates were genetically related. MLVA revealed three clonal complexes in RT027. Six non-RT027 isolates showed four different electrophoretic profiles in PCR ribotyping and were assigned to the MLST sequence types ST2, ST13, ST54, and ST63. The highest discriminatory power showed the genotyping by MLVA. In total, 20 MLVA profiles were identified. This genotyping technique allowed to identify four groups of RT027/ST1 isolates that were indistinguishable by PCR ribotyping and MLST. Our study is the first genotypic characterization of C. difficile isolates in Lithuania. We observed a high prevalence of presumptive RT027 that suggests unfavorable epidemiological situation in Lithuania. Our results stress for implementation of genotyping of C. difficile isolates in Lithuanian surveillance.