The Effect of Three Complexes of Iodine with Amino Acids on Gene Expression of Model Antibiotic Resistant Microorganisms Escherichia coli ATCC BAA-196 and Staphylococcus aureus ATCC BAA-39.

1. BACKGROUND: Iodine is a broad-spectrum antimicrobial disinfectant for topical application. Recent studies have shown promising results on the applicability of an iodine-containing complex, FS-1, against antibiotic-resistant pathogens. It was hypothesized that the antimicrobial activity of iodine-containing complexes may be modulated by the organic moiety of the complex, i.e., amino acids. 2. METHODS: Gene regulation and metabolic alterations were studied in two model multidrug-resistant microorganisms, Staphylococcus aureus ATCC BAA-39, and Escherichia coli ATCC BAA-196, treated with three complexes containing iodine and three different amino acids: glycine, L-alanine, and L-isoleucine. The bacterial cultures were exposed to sub-lethal concentrations of the complexes in the lagging and logarithmic growth phases. Gene regulation was studied by total RNA sequencing and differential gene expression analysis. 3. RESULTS: The central metabolism of the treated bacteria was affected. An analysis of the regulation of genes involved in stress responses suggested the disruption of cell wall integrity, DNA damage, and oxidative stress in the treated bacteria. 4. CONCLUSIONS: Previous studies showed that the application of iodine-containing complexes, such as FS-1, serves as a supplement to common antibiotics and can be a promising way to combat antibiotic-resistant pathogens. Current results shed light on possible mechanisms of this action by disrupting the cell wall barriers and imposing oxidative stress. It was also found that the effect of the complexes on metabolic pathways varied in the tested microorganisms depending on the organic moiety of the complexes and the growth phase when the complexes had been applied.

Component Composition and Biological Activity of Various Extracts of Onosma gmelinii (Boraginaceae).

Onosma roots are widely used in traditional medicine to treat various diseases throughout the world. In this study, for the first time, we investigated the component composition and biological activity of various extracts from the roots of Onosma gmelinii collected in the highlands of the Kakpakty Mountains of the Almaty region (Republic of Kazakhstan). Extracts were obtained by three different methods: percolation extraction, ultrasound-assisted extraction, and supercritical carbon dioxide extraction. The component composition of the extracts was determined by gas chromatography/mass spectrometry (GC/MS), naphthoquinones by thin-layer chromatography (TLC), and spectrophotometric method. In this study, the presence of shikonin and its derivatives in the extracts was confirmed. The concentration of naphthoquinones during CO2 extraction was about 40%, during ultrasonic extraction about 3%, and during percolation extraction about 1.3%. The GC-MS method identified 69 chemical compounds in the ultrasonic extract, 46 compounds in the CO2 extract, and 51 compounds in the percolation extract. The extracts were tested on a panel of bacteria and viruses: two Gram-negative bacteria (Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027); nine Gram-positive bacteria (Staphylococcus aureus ATCC 6538-P, Staphylococcus aureus ATCC BAA-39, Staphylococcus epidermidis ATCC 51625, Staphylococcus epidermidis ATCC 12228, Streptococcus pyogenes ATCC 19615, Streptococcus pneumoniae ATCC BAA-660, Enterococcus hirae ATCC 10541, Enterococcus faecalis ATCC 51575, Enterococcus faecium ATCC 700221); and two fungal species (Candida albicans ATCC 10231, Candida albicans ATCC 2091); five subtypes of influenza virus A (A/FPV/Weybridge/78 (H7N7), A/Swine/Iowa/15/30 (H1N1), A/black-headed gull/Atyrau/743/04 (H13N6), A/FPV/Rostock/1934 (H7N1), A/Almaty/8/98 (H3N2)). The root extracts of Onosma gmelinii showed antibacterial activity in different degrees against all tested Gram-positive bacterial strains, while no inhibitory effect on Gram-negative bacteria was observed. The results indicated that the ultrasonic extract effectively inhibits the growth of the majority of tested Gram-positive bacteria (MBC from 18.3 to 293.0 µg/mL). CO2 extract had the greatest bactericidal activity (MBC from 0.1 to 24.4 µg/mL). Percolation extract insignificantly inhibited bacterial growth (MBC from 2343.8 to 4687.5 µg/mL). CO2 extract and ultrasonic extract significantly reduced the activity of C. albicans. The results of the antiviral action showed that the ultrasonic extract has the greatest effectiveness against different subtypes of the influenza virus A, while other extracts did not show significant activity.

Complete Genome Sequences of Gram-Positive Opportunistic Pathogens Isolated from Hospitals in Almaty, Kazakhstan.

The appearance of drug-resistant pathogens reduces the therapeutic applicability of antibiotics and increases the rate of hospital infections among patients. Complete genome sequences of four Gram-positive clinical isolates of Streptococcus and Staphylococcus were obtained and analyzed to serve as model microorganisms for further studies on drug-induced antibiotic resistance reversion.

Complete Genome Sequences of Gram-Negative Opportunistic Pathogens Isolated in Hospitals in Almaty, Kazakhstan.

The problem of nosocomial infections is growing due to the introduction of new treatment regimens involving immunosuppressive drugs. The genomes of seven Gram-negative clinical isolates of Escherichia, Klebsiella, and Pseudomonas were sequenced and analyzed in this study to serve as model microorganisms to study drug-induced antibiotic resistance reversion.

Transcriptomics and methylomics study on the effect of iodine-containing drug FS-1 on Escherichia coli ATCC BAA-196.

Aim: Promising results on application of iodine-containing nano-micelles, FS-1, against antibiotic-resistant Escherichia coli was demonstrated. Materials & methods: RNA sequencing for transcriptomics and the complete genome sequencing by SMRT PacBio were followed by genome assembly and methylomics. Results & conclusion: FS-1-treated E. coli showed an increased susceptibility to antibiotics ampicillin and gentamicin. Cultivation with FS-1 caused gene expression alterations toward anaerobic respiration, increased anabolism and inhibition of many nutrient uptake systems. Main targets of iodine-containing particles were cell membrane structures causing oxidative, osmotic and acidic stresses. Identification of methylated nucleotides showed an altered pattern in the FS-1-treated culture. Possible role of transcriptional and epigenetic modifications in the observed increase in susceptibility to gentamicin and ampicillin were discussed.

Lay abstract New approaches of combatting drug-resistant infections are in demand as the development of new antibiotics is in a deep crisis. This study was set out to investigate molecular mechanisms of action of new iodine-containing nano-micelle drug FS-1, which potentially may improve the antibiotic therapy of drug-resistant infections. Iodine is one of the oldest antimicrobials and until now there were no reports on development of resistance to iodine. Recent studies showed promising results on application of iodine-containing nano-micelles against antibiotic-resistant pathogens as a supplement to antibiotic therapy. The mechanisms of action, however, remain unclear. The collection strain Escherichia coli ATCC BAA-196 showing an extended spectrum of resistance to ßß-lactam and aminoglycoside antibiotics was used in this study as a model organism. Antibiotic resistance patterns, whole genomes and total RNA sequences of the FS-1-treated (FS) and negative control (NC) variants of E. coli BAA-196 were obtained and analyzed. FS culture showed an increased susceptibility to antibiotics associated with profound gene expression alterations switching the bacterial metabolism to anaerobic respiration, increased anabolism, osmotic stress response and inhibition of many nutrient uptake systems. Nucleotide methylation pattern were identified in FS and NC cultures. While the numbers of methylated sites in both genomes remained similar, some peculiar alterations were observed in their distribution along chromosomal and plasmid sequences.

Chemical Composition and Antimicrobial Activity of Subcritical CO2 Extract of Lepidium latifolium L. (Brassicaceae).

The genus Lepidium L. from Brassicaceae Burnett. family covers over 150 species with an almost cosmopolitan spread. In Kazakhstan, 21 species are described, of which four species are characterized by medicinal properties (L. crassifolium Waldst. et Kit., L. perfoliatum L., L. ruderale L., and L. latifolium L.), used in folk medicine as means of antibacterial, irritant, laxative, antitumor, analgesic, and anthelmintic action. Methods. Raw materials were collected from Almaty region (Republic of Kazakhstan). Lepidium latifolium L. herb's carbon dioxide extract (CO2 extract) was obtained by subcritical carbon dioxide extraction. A gas chromatograph with a mass spectrometric detector was used to determine the component composition of the extract. Antimicrobial activity was determined by two methods: the micromethod of serial dilution and the disc-diffusion method. Four microbial test strains were used: Staphylococcus aureus ATCC 6538-P, Escherichia coli ATCC 8739, Klebsiella pneumonia ATCC 10031, and Candida albicans ATCC 10231. Results. The technology of carbon dioxide extraction has undoubted advantages over traditional methods of extraction: it has a controlled selectivity in relation to groups of biologically active substances, allows deep extraction, and maximizes the release of rich complexes of compounds contained in plants. In this study, firstly, the CO2 extract was obtained under subcritical conditions from the aerial part of L. latifolium L., and the composition was determined. Hexane was the best solvent for CO2 extract, and 40 components were identified. Screening of antimicrobial activity of the L. latifolium's CO2 extract showed the essential activity of all clinically significant strains tested: Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, and Candida albicans. Conclusions. This research showed that the CO2 extract of the raw material of Lepidium latifolium L. contains biologically active compounds exhibiting an essential antimicrobial effect, and therefore it is possible to recommend for the development of various drugs for use in medical practice.

The Effect of Iodine-Containing Nano-Micelles, FS-1, on Antibiotic Resistance, Gene Expression and Epigenetic Modifications in the Genome of Multidrug Resistant MRSA Strain Staphylococcus aureus ATCC BAA-39.

Application of supplementary drugs which increase susceptibility of pathogenic bacteria to antibiotics is a promising yet unexplored approach to overcome the global problem of multidrug-resistant infections. The discovery of a new drug, an iodine-containing nano-molecular complex FS-1, which has proven to improve susceptibility to antibiotics in various pathogens, including MRSA strain Staphylococcus aureus ATCC BAA-39TM, allowed studying this phenomenon. Chromosomal DNA and total RNA samples extracted from the FS-1 treated strain (FS) and from the negative control (NC) cultures were sequenced by PacBio SMRT and Ion Torrent technologies, respectively. PacBio DNA reads were used to assemble chromosomal DNA of the NC and FS variants of S. aureus BAA-39 and to perform profiling of epigenetically modified nucleotides. Results of transcriptional profiling, variant calling and detection of epigenetic modifications in the FS variant were compared to the NC variant. Additionally, the genetic alterations caused by the treatment of S. aureus BAA-39 with FS-1 were compared to the results of a similar experiment conducted with another model organism, E. coli ATCC BAA-196. Several commonalities in responses of these phylogenetically distant microorganisms to the treatment with FS-1 were discovered, which included metabolic transition toward anaerobiosis and oxidative/osmotic stress response. S. aureus culture appeared to be more sensitive to FS-1 due to a higher penetrability of cells by iodine bound compounds, which caused carbonyl stress associated with nucleotide damaging by FS-1, abnormal epigenetic modifications and an increased rate of mutations. It was hypothesized that the disrupted pattern of adenine methylated loci within methicillin-resistance chromosome cassettes (SCCmec) may promote excision of this antibiotic resistance determinant from chromosomes while the altered pattern of cytosine methylation was behind the adaptive gene regulation in the culture FS. The selection against the antibiotic resistance in bacterial populations caused by abnormal epigenetic modifications exemplifies possible mechanisms of antibiotic resistance reversion induced by iodine-containing compounds. These finding will facilitate development of therapeutic agents against multidrug-resistant infections.

Complete Genome Sequence of Collection Strain Acinetobacter baumannii ATCC BAA-1790, Used as a Model To Study the Antibiotic Resistance Reversion Induced by Iodine-Containing Complexes.

The strain Acinetobacter baumannii ATCC BAA-1790 was sequenced as a model for nosocomial multidrug-resistant infections. Long-read PacBio sequencing revealed a circular chromosome of 3,963,235 bp with two horizontally transferred genomic islands and a 67,023-bp plasmid. Multiple antibiotic resistance genes and genome methylation patterns were identified.

Complete Genome Sequence of a Multidrug-Resistant Strain, Escherichia coli ATCC BAA-196, as a Model for Studying Induced Antibiotic Resistance Reversion.

Here, we report the complete genome sequence of the multidrug-resistant Escherichia coli strain ATCC BAA-196, a model organism used for studying possible antibiotic resistance reversion induced by FS-1, an iodine-containing complex. Two genomes, representing FS-1-treated and negative-control variants and composed of a chromosome and several plasmids, were assembled.

Assembly of Complete Genome Sequences of Negative-Control and Experimental Strain Variants of Staphylococcus aureus ATCC BAA-39 Selected under the Effect of the Drug FS-1, Which Induces Antibiotic Resistance Reversion.

Staphylococcus aureus ATCC BAA-39 is the reference organism for a multidrug-resistant Staphylococcus aureus (MRSA) strain that was used to study drug-induced resistance reversion by an iodine-containing nanomolecular complex, FS-1. PacBio sequencing was performed on both the experimental and control strains, followed by genome assembly, variant calling, and DNA modification profiling.