Development of Resistance to Clarithromycin and Amoxicillin-Clavulanic Acid in Lactiplantibacillus plantarum In Vitro Is Followed by Genomic Rearrangements and Evolution of Virulence.

Ensuring the safety of the use of probiotics is a top priority. Obviously, in addition to studying the beneficial properties of lactic acid bacteria, considerable attention should be directed to assessing the virulence of microorganisms as well as investigating the possibility of its evolution under conditions of selective pressure. To assess the virulence of probiotics, it is now recommended to analyze the genomes of bacteria in relation to the profiles of the virulome, resistome, and mobilome as well as the analysis of phenotypic resistance and virulence in vitro. However, the corresponding procedure has not yet been standardized, and virulence analysis of strains in vivo using model organisms has not been performed. Our study is devoted to testing the assumption that the development of antibiotic resistance in probiotic bacteria under conditions of selective pressure of antimicrobial drugs may be accompanied by the evolution of virulence. In this regard, special attention is required for the widespread in nature commensals and probiotic bacteria actively used in pharmacology and the food industry. As a result of step-by-step selection from the Lactiplantibacillus plantarum 8p-a3 strain isolated from the "Lactobacterin" probiotic (Biomed, Russia), the L. plantarum 8p-a3-Clr-Amx strain was obtained, showing increased resistance simultaneously to amoxicillin-clavulanic acid and clarithromycin (antibiotics, the combined use of which is widely used for Helicobacter pylori eradication) compared to the parent strain (MIC8p-a3-Clr-Amx of 20 µg/mL and 10 µg/mL, and MIC8p-a3 of 0.5 µg/mL and 0.05 µg/mL, respectively). The results of a comparative analysis of antibiotic-resistant and parental strains indicate that the development of resistance to the corresponding antimicrobial drugs in L. plantarum in vitro is accompanied by the following: (i) significant changes in the genomic profile (point mutations as well as deletions, insertions, duplications, and displacement of DNA sequences) associated in part with the resistome and mobilome; (ii) changes in phenotypic sensitivity to a number of antimicrobial drugs; and (iii) an increase in the level of virulence against Drosophila melanogaster, a model organism for which L. plantarum is considered to be a symbiont. The data obtained by us indicate that the mechanisms of adaptation to antimicrobial drugs in L. plantarum are not limited to those described earlier and determine the need for comprehensive studies of antibiotic resistance scenarios as well as the trajectories of virulence evolution in probiotic bacteria in vivo and in vitro to develop a standardized system for detecting virulent strains of the corresponding microorganisms. IMPORTANCE Ensuring the safety of the use of probiotics is a top priority. We found that increased resistance to popular antimicrobial drugs in Lactiplantibacillus plantarum is accompanied by significant changes in the genomic profile and phenotypic sensitivity to a number of antimicrobial drugs as well as in the level of virulence of this bacterium against Drosophila. The data obtained in our work indicate that the mechanisms of antibiotic resistance in this bacterium are not limited to those described earlier and determine the need for comprehensive studies of the potential for the evolution of virulence in lactic acid bacteria in vivo and in vitro and to develop a reliable control system to detect virulent strains among probiotics.

Adaptation to Antimicrobials and Pathogenicity in Mycoplasmas: Development of Ciprofloxacin-Resistance and Evolution of Virulence in Acholeplasma laidlawii.

For the first time it was shown that the development of resistance to ciprofloxacin in vitro in Acholeplasma laidlawii, a mycoplasma which is widely spread in nature and which is the main contaminant of cell cultures and vaccines, is associated with diverse pathways of virulence evolution: virulome and virulence differ significantly between ciprofloxacin-resistant strains, including those with the same level of antimicrobial resistance.

Extracellular Vesicles from Mycoplasmas Can Penetrate Eukaryotic Cells In Vitro and Modulate the Cellular Proteome.

The extracellular vesicles (EVs) produced by bacteria transport a wide range of compounds, including proteins, DNA and RNA, mediate intercellular interactions, and may be important participants in the mechanisms underlying the persistence of infectious agents. This study focuses on testing the hypothesis that the EVs of mycoplasmas, the smallest prokaryotes capable of independent reproduction, combined in the class referred to as Mollicutes, can penetrate into eukaryotic cells and modulate their immunoreactivity. To verify this hypothesis, for the first time, studies of in vitro interaction between human skin fibroblasts and vesicles isolated from Acholeplasma laidlawii (the ubiquitous mycoplasma that infects higher eukaryotes and is the main contaminant of cell cultures and vaccines) were conducted using confocal laser scanning microscopy and proteome profiling, employing a combination of 2D-DIGE and MALDI-TOF/TOF, the Mascot mass-spectrum analysis software and the DAVID functional annotation tool. These studies have revealed for the first time that the extracellular vesicles of A. laidlawii can penetrate into eukaryotic cells in vitro and modulate the expression of cellular proteins. The molecular mechanisms behind the interaction of mycoplasma vesicles with eukaryotic cells and the contribution of the respective nanostructures to the molecular machinery of cellular permissiveness still remain to be elucidated. The study of these aspects is relevant both for fundamental research into the "logic of life" of the simplest prokaryotes, and the practical development of efficient control over hypermutable bacteria infecting humans, animals and plants, as well as contaminating cell cultures and vaccines.

Adaptation of Mycoplasmas to Antimicrobial Peptides: Development of Melittin Resistance in Acholeplasma laidlawii Is Associated with Changes in Genomic and Proteomic Profiles and in Virulence.

For the first time it is shown that the development of resistance to melittin in Acholeplasma laidlawii, a mycoplasma that is widely spread in nature and that is the main contaminant of cell cultures and vaccines, is associated with significant changes in the genomic profile, in cellular and vesicular proteomes, as well as in virulence.

Data on proteomic profiling of cells and extracellular vesicles of the melittin-resistant Acholeplasma laidlawii strain.

Acholeplasma laidlawii (class Mollicutes), a major contaminant of cell cultures, quickly adapts to various classes of antimicrobials, including antimicrobial peptides. The extracellular vesicles of this bacterium can play a significant role in the development of drug-resistance Chernov et al., 2018. We compared the cellular and vesicular proteomes of A. laidlawii strains with differing susceptibility to melittin (an antimicrobial peptide from bee venom), the genomes of which we have previously sequenced. We extracted soluble proteins from cells and extracellular vesicles of the A. laidlawii PG8RMel strain showing an increased resistance to melittin, and compared them with the cellular proteome and a previously obtained vesicular proteome of the original (reference) A. laidlawii PG8B strain Chernov et al., 2014. The cellular proteome profile of the A. laidlawii strains differing in susceptibility to melittin was determined by using two-dimensional gel electrophoresis and MALDI-TOF/TOF MS. Here we present the cellular proteins that were differentially expressed. The vesicular proteome profile was determined by using one-dimensional electrophoresis and chromatography-mass spectrometry. A list of the extracellular vesicles proteins of the melittin-resistant A. laidlawii strain is presented here.

Mycoplasmas and Novel HO-1 Inducers: Recent Advances.

Inflammation and the ways for its regulation: The development of an effective system for the treatment of inflammatory diseases requires comprehensive studies of the cellular signaling molecular networks comprising responses to various stressors, including pathogenic and non-pathogenic microorganisms. Significant attention on fundamental and applied research has recently focused on inducers of hemе oxygenase-1 (HO-1) and inhibitors of the expression of this enzyme, which regulates expression of this and other cytoprotective molecules and modulation of inflammation. Recent studies indicate that mycoplasmas (a major group of human pathogens of the Mollicutes) are capable of modulating inflammatory responses through the activation of the Nrf2 and the expression of HO-1. In vitro experiments demonstrate that the membrane lipoproteins (LAMPs), along with lipoprotein derivatives (lipopeptide MALP-2) in mycoplasmas cause a "cross-talk" between the pro- and antiinflammatory signaling pathways. Importantly, lipopeptide/lipoprotein - induced expression of HO-1 tends to suppress inflammation. Conclusion: The study of the molecular network that causes the corresponding outcome can facilitate the development of new approaches for the treatment of inflammatory processes. The derivatives of LAMPs and MALP-2 and of their analogues may prove promising for the treatment of diseases associated with chronic inflammation.

Mycoplasmas and Their Antibiotic Resistance: The Problems and Prospects in Controlling Infections.

The present review discusses the problem of controlling mycoplasmas (class Mollicutes), the smallest of self-replicating prokaryotes, parasites of higher eukaryotes, and main contaminants of cell cultures and vaccines. Possible mechanisms for the rapid development of resistance to antimicrobial drugs in mycoplasmas have been analyzed. Omics technologies provide new opportunities for investigating the molecular basis of bacterial adaptation to stress factors and identifying resistomes, the total of all genes and their products contributing to antibiotic resistance in microbes. The data obtained using an integrated approach with post-genomics methods show that antibiotic resistance may be caused by more complex processes than has been believed heretofore. The development of antibiotic resistance in mycoplasmas is associated with essential changes in the genome, proteome, and secretome profiles, which involve many genes and proteins related to fundamental cellular processes and virulence.

Genomic and proteomic profiles of Acholeplasma laidlawii strains differing in sensitivity to ciprofloxacin.

As a result of comparative analysis of complete genomes as well as cell and vesicular proteomes of A. laidlawii strains differing in sensitivity to ciprofloxacin, it was first shown that the mycoplasma resistance to the antibiotic is associated with the reorganization of genomic and proteomic profiles, which concerns many genes and proteins involved in fundamental cellular processes and realization of bacterial virulence.

Mycoplasma Contamination of Cell Cultures: Vesicular Traffic in Bacteria and Control over Infectious Agents.

Cell cultures are subject to contamination either with cells of other cultures or with microorganisms, including fungi, viruses, and bacteria. Mycoplasma contamination of cell cultures is of particular importance. Since cell cultures are used for the production of vaccines and physiologically active compounds, designing a system for controlling contaminants becomes topical for fundamental science and biotechnological production. The discovery of extracellular membrane vesicles in mycoplasmas makes it necessary to take into consideration the bacterial vesicular traffic in systems designed for controlling infectious agents. The extracellular vesicles of bacteria mediate the traffic of proteins and genes, participate in cell-to-cell interactions, as well as in the pathogenesis and development of resistance to antibiotics. The present review discusses the features of mycoplasmas, their extracellular vesicles, and the interaction between contaminants and eukaryotic cells. Furthermore, it provides an analysis of the problems associated with modern methods of diagnosis and eradication of mycoplasma contamination from cell cultures and prospects for their solution.

[Ribonucleolytic activity of mycoplasmas].

Mycoplasmas are incapable of de novo synthesis of nucleotides and must therefore secrete nucleases in order to replenish the pool of nucleic acid precursors. The nucleolytic activity of mycoplasmas is an important factor in their pathogenicity. Bacterial ribonucleases (RNases) may produce a broad spectrum of biological effects, including antiviral and antitumor activity. Mycoplasma RNases are therefore of interest. In the present work, capacity of Acholeplasma laidlawii and Mycoplasma hominis for RNase synthesis and secretion was studied. During the stationary growth phase, these organisms were found to synthesize Mg(2+)-dependent RNases, with their highest activity detected outside the cells. Localization of A. laidlawii RNases was determined: almost 90% of the RNase activity was found to be associated with the membrane vesicles. Bioinformational analysis revealed homology between the nucleotide sequences of 14 Bacillus subtilis genes encoding the products with RNase activity and the genes of the mycoplasmas under study. Amino acid sequences of 4 A. laidlawii proteins with ribonuclease activity and the Bsn RNase was also established.

[Manganese in atherogenesis: detection, origin, and role].

The role of transition metal ions in atherogenesis is controversial; they can participate in the hydroxyl radical generation and catalyze the reactive oxygen species neutralization reaction as cofactors of antioxidant enzymes. Using EPR spectroscopy, we revealed that 70% of the samples of aorta with atherosclerotic lesions possessed superoxide dismutase activity, 100% of the samples initiated Fenton reaction and demonstrated the presence of manganese paramagnetic centers. The sodA gene encoding manganese-dependent bacterial superoxide dismutase was not found in the samples of atherosclerotic plaques by PCR using degenerate primers. The data obtained indicates the perspectives of manganese analysis as a marker element in the express diagnostics of atherosclerosis.

[The adaptation of mycoplasmas to stress conditions: features of proteome shift in Mycoplasma hominis PG37 under starvation and low temperature].

Mycoplasma hominis--one of the widely spread mycoplasmas (class Mollicutes), associated with the socially significant human diseases and contamination of cell cultures. The solution of the problem on controlling M. hominis infections is connected with determination of the molecular basis, responsible for mechanisms of bacterium survival under unfavorable conditions. As a result of proteomic approach (2-DIGE and MALDI TOF/TOF MS) for the first time, 53 M. hominis PG37 proteins were detected, different abundance of which occurred at cultivating the bacterium under stress (starvation and low temperature) conditions. According to the classification of proteins by functional category (clusters of orthologous groups of proteins--COG), 47 of the 53 proteins of the mycoplasma are involved in the fundamental cellular and biochemical processes--translation (12; 22.64%), transcription (2; 3.77%), posttranslational modification (7; 13.20%), cell cycle control (2; 3.77%), energy production and conversion (6; 11.32%), carbohydrate transport and metabolism (3; 5.66%), amino acid transport and metabolism (8; 15.09%), nucleotide transport and metabolism (6; 11.32%), inorganic ion transport and metabolism (1; 1.89%). The functions of six proteins (11.32%) have not been found; 24 proteins (45.28%) are the factors of bacterium virulence. M. hominis PG37 proteins, the expression modulation of which arises under the unfavorable environmental conditions, are the components of adaptation mechanisms of the mycoplasma to the stressors and potential targets for controlling infections caused by this bacterium.

[Adaptation of mycoplasmas to stressful factors: nucleotide sequences nonregistered in vegetative forms of M. galisepticum S6 cells are revealed in nonculturable forms of the mycoplasma].

Adaptation of M. gallisepticum S6 to unfavorable environmental conditions is connected with transformation of the vegetative forms of the mycoplasma cells to the viable but non-culturable (VBNC) forms. The vegetative forms and the VBNC forms differ in the spectrum of the PCR-products that was forming due to amplification of the nucleotide sequence of the pvpA-gene coding the able cytoadhesion protein. As to vegetative forms of the mycoplasma the only amplicon, containing one open reading frame (1086 b.p.) with a high homology (97%) to the pvpA-gene of M. gallisepticum R and Pendik is detected. In the case of VBNC forms of M. gallisepticum S6, the additional amplicons besides those indicating the pvpA-gene of the mycoplasma are observed. In the nucleotide sequences of the additional amplicons, the open reading frames are detected that are not registered in the database of the complete sequence of the mycoplasma genome. A high homology (54-55%) of the nucleotide sequences of the pvpA-gene and the additional pvpA-amplicons allows to suggest that thepvpA-gene sequence seems to be a basis for forming new regions within the mycoplasma genome during adaptation of the bacterium to unfavorable environmental conditions.