Background/Objectives: Tuberculosis (TB) remains the leading cause of death from a single infectious agent worldwide. In line with the World Health Organization’s (WHO) goal to end TB by 2035, the rapid development and clinical implementation of new, effective vaccines is urgently needed. To support global TB control efforts, we developed a novel candidate subunit multistage vaccine. Methods: This vaccine incorporates multiple Mycobacterium tuberculosis antigens expressed during both dormant and active stages of infection, fused into a single recombinant protein (ESAT6-CFP10-Ag85A-Rv2660c-Rv1813c). The antigen was encapsulated in biodegradable poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles along with the pattern recognition receptor (PRR) agonists monophosphoryl lipid A (MPLA) and muramyl dipeptide (MDP), which function as adjuvants. Results: Using a mixed intramuscular/nasal prime-boost regimen, the vaccine elicited a mixed Th1/Th17 cell-mediated immune response, as well as a robust humoral response characterized by sustained systemic IgG (lasting at least one year) and prominent local secretory IgA. The vaccine demonstrated protective efficacy as a prophylactic booster following BCG priming in both murine and guinea pig models and was also effective in a therapeutic setting in a murine infection model. Conclusions: The results of this study provide empirical evidence that multistage tuberculosis vaccines represent a promising strategy for achieving global TB control.

Residents of the International Space Station (ISS) are various types of bacteria that can act as potential biodegraders of structural materials, which entails disruptions in the operation of various equipment and, indirectly, provoke medical risks and affect the health of crew members with reduced immunity. Since the beginning of ISS exploitation sanitary and microbiological monitoring of the habitat has been carried out for the purpose of timely detection and inactivation of potentially dangerous strains. The study was conducted to investigate the dynamics of the composition and number of bacterial strain species detected on the internal surfaces of the ISS interior and equipment for the period covering from Expedition 46 to Expedition 67 (2016–2022). Samples collected from ISS interior surfaces were grown on nutrient media. Strains were identified by 16S RNA sequencing and MALDI-TOF analysis. The results obtained showed that the number of bacterial community species increases with the increase of the ISS RS operation period. However, this growth was not linear, but was a wave-like repetitive process of changing phases of activation and stagnation of the microbiome. During the period of work of Expeditions 46-67, species of the genera Bacillus and Staphylococcus dominated in the microbiome. Among representatives of the genus Bacillus, the species B. licheniformis dominated. The predominance of bacteria of this genus, apparently, is due to their high resistance to physical and chemical parameters of the ISS habitat, including DNA-damaging factors, such as increased radiation background, which, in turn, can be explained by their ability to spore formation. The results obtained once again indicate the high evolutionary plasticity of B. licheniformis strains, which we discovered earlier in other studies.

BACKGROUND:Protease S (PrtS) from Photorhabdus laumondii belongs to the group of protealysin-like proteases (PLPs), which are understudied factors thought to play a role in the interaction of bacteria with other organisms. Since P. laumondii is an insect pathogen and a nematode symbiont, the analysis of the biological functions of PLPs using the PrtS model provides novel data on diverse types of interactions between bacteria and hosts.METHODS AND RESULTS:Recombinant PrtS was produced in Escherichia coli. Efficient inhibition of PrtS activity by photorin, a recently discovered emfourin-like protein inhibitor from P. laumondii, was demonstrated. The Galleria mellonella was utilized to examine the insect toxicity of PrtS and the impact of PrtS on hemolymph proteins in vitro. The insect toxicity of PrtS is reduced compared to protease homologues from non-pathogenic bacteria and is likely not essential for the infection process. However, using proteomic analysis, potential PrtS targets have been identified in the hemolymph.CONCLUSIONS:The spectrum of identified proteins indicates that the function of PrtS is to modulate the insect immune response. Further studies of PLPs' biological role in the PrtS and P. laumondii model must clarify the details of PrtS interaction with the insect immune system during bacterial infection.

Primary ciliary dyskinesia (PCD) is a rare hereditary disease belonging to the group of ciliopathies. PCD is caused by a defect in the ultrastructure of the cilia of the respiratory epithelium and similar structures (sperm flagella, fallopian tube villi, ventricular ependyma) with a prevalence of 1:10,000 to 1:30,000 people. One of the main problems associated with this condition is frequent recurrent bacterial infections, which lead to decreased lung function and the development of bronchiectasis. The aim was to study the microbial landscape of the respiratory tract of patients with PCD. Methods. We studied the medical history and bacteriological assays of 170 throat, nasal, and sputum samples from 64 patients diagnosed with PCD. Bacteriological methods, RT-PCR, and MALDI-TOF mass spectrometry were used to identify microorganisms. Results. The bacteriological assay identified 46 bacterial species, 4 viral species, and representatives of 2 fungal genera. The predominant bacteria were Staphylococcus aureus (62.5%) and Pseudomonas aeruginosa (26.6%). The identified fungi were Candida spp. and Aspergillus spp. Viruses (Adenoviridae, Coronaviridae, Rhinoviridae) were detected in 7 patients in association with representatives of normal microflora and clinically significant microorganisms such as S. aureus, Klebsiella pneumoniae, P. aeruginosa. Achromobacter spp., Stenotrophomonas maltophilia, Acinetobacter spp., and Moraxella spp. were isolated in individual cases in patients of all age groups. Conclusion. The respiratory microflora was characterized by a species diversity of pathogens and was mixed in 79.7% of patients. A viral-bacterial associations was observed in 10.9% of patients. S. aureus was the predominant pathogen in the group of children and adolescents (58.3% and 82.6%, respectively), whereas P. aeruginosa predominated (52.9%) in the adult patients.

Bacillus cereus sensu lato comprises genetically, morphologically, and physiologically similar gram-positive spore-forming bacterial species with high pathogenic potential, such as B. anthracis, B. cereus, and B. thuringiensis. Toxin-producing strains of B. cereus s.I. pose a major threat to human health. The high degree of similarity between these species makes it very difficult to identify them and to take adequate measures to treat the diseases they cause. Previously, we characterized the clinical isolate CCGC 19/16 belonging to B. cereus s.l. that exhibited features of both B. cereus and B. cytotoxicus. In the present work, CCGC 19/16 was identified as B. cytotoxicus using multilocus sequence typing (MLST) and mass spectrometric analysis. It was also shown that, unlike other representatives of the B. cytotoxicus species, strain CCGC 19/16 is not thermotolerant. Unlike B. cereus, strain CCGC 19/16 is sensitive to most antibiotics and shows increased motility. Like B. cereus strain CCGC 19/16 forms β-hemolysis zones in blood agar. In addition, it has been shown that prolonged storage of samples prior to analysis can lead to misidentification of the isolate. Our results indicate that "rapid methods" of analysis using single genes have insufficient resolving power in the identification of B. cereus s.l. species. The combination of MLST analysis with MALDI-TOF MS provides sufficient resolution.

MALDI-TOF MS represents a rapid and cost-effective method for identifying proteins and microorganisms. When obtaining recombinant protein producers, differences in expression levels among transformants necessitate the conduction of a small-scale screening of expression levels. This study proposes a fast and easy method for screening clones producing recombinant proteins using MALDI-TOF MS. Various recombinant proteins were utilized to test the proposed method ### Competing Interest Statement The authors have declared no competing interest.

The main cause of death in patients with cystic fibrosis (CF) is infectious process in the lungs, in particular, chronic lung infections caused by various pathogens, most often a combination of bacteria, fungi, or viruses. Data on mixed bacterial and viral-bacterial infections from domestic and foreign sources are fragmentary and sparse. The dominant associations of bacterial and viral pathogens in patients with cystic fibrosis have not been studied properly, and data on their epidemiological significance are lacking. The aim of this study was to assess the prevalence of bacterial and viral infections in patients with cystic fibrosis and to substantiate the need for the development of virological monitoring. Methods . Biomaterials from the respiratory tract of CF patients (409 children and 160 adults with CF) examined from 2006 to 2022 were used. The study was carried out using bacteriological methods, molecular genetic methods (RT-PCR) and MALDI-TOF mass-spectrometry. Results . Microflora of the respiratory tract was shown to be mixed in 2/3 patients with CF. The microflora of the lungs of children with CF is a dynamic community of microorganisms with high diversity and variability. In adult patients, associations of microorganisms are more common than in children, but the composition of associations is less diverse. We isolated about 40 species of bacteria from adult patients and more than 85 species from children in our sample. NFMO prevailed, including Burkholderia cepacia complex, Pseudomonas aeruginosa, Achromobacter xylosoxidans, Achromobacter ruhlandii, Stenotrophomonas maltophilia, and Staphylococcus aureus, Candida albicans, Aspergillus spp . Real-time PCR showed the presence of rhinovirus RNA in 10% of samples obtained from children and 12.9% from adults with cystic fibrosis. Conclusion . Our results indicate the need for continuous monitoring of the lung microflora in patients with CF, including testing for viruses.

— Bacillus cereus is a spore-forming bacterium found in the environment mainly in soil. Bacillus spores are known to be extremely resistant not only to environmental factors, but also to various sanitation regimes. This leads to spore contamination of toxin-producing strains in hospital and food equipment and, therefore, poses a great threat to human health. Two clinical isolates identified as B. cereus and B. cytotoxicus were used in the present work. It was shown that their calcium ion content was significantly lower than that of the reference strains. According to electron microscopy, one of the SRCC 19/16 isolates has an enlarged exosporium, and the SRCC 1208 isolate has large electron-dense inclusions of an unclear nature during sporulation. We can assume that these contain a biologically active component with a cytotoxic effect and possibly play a role in pathogenesis. Comparative chemical, biochemical, physiological, and ultrastructural analysis of spores of clinical isolates and reference strains of B. cereus was performed. The results we obtained deepen our understanding of the properties of spores that contribute to the increased pathogenicity of B. cereus group species.

Endopeptidases IdeS and IdeZ (streptococcal virulence factors that specifically cleave IgG heavy chains) are of particular interest because of their potential use in biotechnology, medicine, and veterinary. Genes encoding these enzymes were cloned and expressed in Escherichia coli heterologous expression system ( ideS was cloned from a Streptococcus pyogenes collection strain; ideZ from Streptococcus zooepidemicus was synthesized). The 6His-tag was introduced into the amino acid sequence of each endopeptidase, and IdeS and IdeZ were purified by metal affinity chromatography to an apparent homogeneity (according to polyacrylamide gel electrophoresis). Purified enzymes were active against human and animal IgGs; their specificity toward human IgGs was confirmed by polyacrylamide gel electrophoresis. Recombinant IdeZ was used for immunological analysis of equine strangles infection (diagnostics and determination of the titer of specific antibodies in blood). Hence, IdeZ can be used in veterinary and sanitary microbiology to diagnose infections caused by Streptococcus equi and S. zooepidemicus in addition to its application in medicine and biotechnology.

The study aimed to establish the role of the home environment as a source of Pseudomonas aeruginosa infection in patients with cystic fibrosis.

We studied the effect of the L. plantarum strain supernatant on the growth of culture and biofilm of non-fermenting bacteria of the genera Pseudomonas, Achromobacter, and Burkholderia. To obtain a supernatant, the culture of L. plantarum was grown for 48 h at 37°C on a Lactic broth nutrient medium with casein peptone, then centrifuged and filtered through a 0.22-μm Millipore filter. Antimicrobial activity was determined by broth microdilution assay. The inhibitory effect of the supernatant on the growth of bacteria of all three genera was demonstrated. The maximum inhibition was observed for P. aeruginosa (by 13 times compared to the control). For bacteria of the Achromobacter and Burkholderia genera, the inhibition was less pronounced: by 7 and 6 times, respectively. The supernatant also inhibited biofilm formation by P. aeruginosa and A. ruhlandii, but did not affect formed biofilm. Thus, the L. plantarum supernatant obtained by us exhibited pronounced antimicrobial activity against non-fermenting bacteria, the causative agents of nosocomial infections, especially in immunocompromised individuals, very often in cystic fibrosis patients.

Objective. To provide a rationale for microbiological monitoring of chronic lung infection caused by Achromobacter spp. in patients with cystic fibrosis (CF) to develop an adequate preventive and therapeutic strategy. Materials and methods. This study included 142 Achromobacter strains isolated from 55 children and 35 adults with CF during their follow-up. In this study, classical microbiological (culture) and modern molecular genetic methods (polymerase chain reaction (PCR), multilocus sequence typing (MLST) and whole-genome sequencing (WGS)), and MALDI-TOF-MS were used. Results. Among the examined patients with chronic lung infection caused by bacteria from the genus Achromobacter, monoinfection was detected in 5.8% of patients. In other cases, associations of Achromobacter spp. with other bacterial species were observed. A. ruhlandii (76%) and A. xylosoxidans (7.2%) were the most frequently isolated species. It was shown that the microbiota in CF patients with chronic lung infection caused by Achromobacter spp. is characterized by variability and is related to long-term circulation of both one genotype with different subpopulation phenotypes and circulation of 2 or more genotypes or species of Achromobacter spp., which has clinical and epidemiological significance. Conclusion. Successful prevention and treatment of Achromobacter spp.-associated infections in CF patients require continuous microbiological monitoring of chronic infection, including that of phenotypic and genotypic properties of Achromobacter spp. strains isolated from patients. Key words: chronic lung infection, Achromobacter sp., microbiological monitoring, genotypic and phenotypic heterogeneity, sensitivity to antibiotics

Spore-forming bacteria are residents on the International space station (ISS) and can act as potential biodistructors of structural materials with a consequent disturbance of equipment operation. These bacteria demonstrate high resistance to many stress factors. Molecular mechanisms of this resistance are poorly understood. Sanitary/hygienic monitoring resulted in detection and isolation of strain Bacillus pumilus 25. The investigation was aimed to test B. pumilus 25 resistance to oxidative agents, antibiotics, and to analyze expression of genes associated with the cell response to DNA damage and hydrogen sulfide production. The strain was identified by sequencing 16S RNA and MALDI-TOF analysis. Resistance to stresses and antibiotics was tested with the use of standard microbiological methods. Level of mRNA genes was determined with the real-time polymerase chain reaction. Our results evidence for heightened B. pumilus 25 resistance to the oxidative stress and majority of the antibiotics used in the investigation. This resistance seems to be associated with high expression of the ku-ligD operon genes, and elevated production of hydrogen sulfide.

Extracellular matrix plays a pivotal role in biofilm biology and proposed as a potential target for therapeutics development. As matrix is responsible for some extracellular functions and influence bacterial cytotoxicity against eukaryotic cells, it must have unique protein composition. P. aeruginosa is one of the most important pathogens with emerging antibiotic resistance, but only a few studies were devoted to matrix proteomes and there are no studies describing matrix proteome for any clinical isolates except reference strains PAO1 and ATCC27853. Here we report the first biofilm matrix proteome of P. aeruginosa isolated from bronchoalveolar lavage of patient in intensive care unit. We have identified the largest number of proteins in the matrix among all published studies devoted to P. aeruginosa biofilms. Comparison of matrix proteome with proteome from embedded cells let us to identify several enriched bioprocess groups. Bioprocess groups with the largest number of overrepresented in matrix proteins were oxidation-reduction processes, proteolysis, and transmembrane transport. The top three represented in matrix bioprocesses concerning the size of the GO annotated database were cell redox homeostasis, nucleoside metabolism, and fatty acid synthesis. Finally, we discuss the obtained data in a prism of antibiofilm therapeutics development.

Extracellular matrix plays a pivotal role in biofilm biology and proposed as a potential target for therapeutics development. As matrix is responsible for some extracellular functions and influence bacterial cytotoxicity against eukaryotic cells, it must have unique protein composition. P. aeruginosa is one of the most important pathogens with emerging antibiotic resistance, but only a few studies were devoted to matrix proteomes and there are no studies describing matrix proteome for any clinical isolates. Here we report the first biofilm matrix proteome of P. aeruginosa isolated from bronchoalveolar lavage of patient in intensive care unit. We have identified the largest number of proteins in the matrix among all published studies devoted to P. aeruginosa biofilms. Comparison of matrix proteome with proteome from embedded cells let us to identify several enriched bioprocess groups. Bioprocess groups with the largest number of overrepresented in matrix proteins were oxidation-reduction processes, proteolysis, and transmembrane transport. The top three represented in matrix bioprocesses concerning the size of the GO annotated database were cell redox homeostasis, nucleoside metabolism, and fatty acid synthesis. Finally, we discuss the obtained data in a prism of antibiofilm therapeutics development.

Microsporum canis is an important fungal pathogen that is mainly transmitted to humans from domestic animals. Different strains of M. canis vary in their ability to infect humans. Comparative studies of M. canis strains from humans and animals using MALDI mass spectrometry have not yet been published. In a comparative aspect, M. canis strains isolated from human (HS 09-18) and cat (FC 19-18) were studied. Comparison of the obtained mass spectra revealed that M. canis HS 09-18 displayed characteristic peaks with m/z 3339, 4287, 6485, 7740 and 8568, while M. canis FC 19-18 displayed unique peaks with m/z 5192, 7221 and 8049. Three peaks with maximum intensity with m/z 6112, 6828, 9625 were found in both M. canis strains. The dendrogram built on the basis of MALDI mass spectra revealed the clustering of M. canis strains in accordance with their origin: strains from cats and humans formed two separate clusters. Thus, clear differences were demonstrated between M. canis strains depending on the source of origin (cat and human). MALDI mass spectrometry makes it possible to carry out species identification, as well as to reveal intraspecific differences in M. canis. The mechanisms that allow dermatophytes to switch from zoophilic to anthropophilic behavior need to be further explored.

Chromobacterium species are common in tropical and subtropical zones in environmental samples according to numerous studies. Here, we describe an environmental case of resident Chromobacterium vaccinii in biofilms associated with Carex spp. roots in Moscow region, Russia (warm-summer humid continental climate zone). We performed broad characterization of individual properties as well as surrounding context for better understanding of the premise of C. vaccinii survival during the winter season. Genome properties of isolated strains propose some insights into adaptation to habit and biofilm mode of life, including social cheaters carrying ΔluxR mutation. Isolated C. vaccinii differs from previously described strains in some biochemical properties and some basic characteristics like fatty acid composition as well as unique genome features. Despite potential to modulate membrane fluidity and presence of several genes responsible for cold shock response, isolated C. vaccinii did not survive during exposure to 4 °C, while in the complex biofilm sample, it was safely preserved for at least half a year in vitro at 4 °C. The surrounding bacterial community within the same biofilm with C. vaccinii represented a series of psychrophilic bacterial species, which may share resistance to low temperatures with other species within biofilm and provide C. vaccinii an opportunity to survive during the cold winter season.