21 Recent studies have demonstrated that antibiotic resistance in pathogenic bacteria could be 22 reverted to the drug sensitive phenotype by the treatment with the newly introduced iodine 23 containing nanomolecular complex, FS-1. Antibiotic resistance reversion has been verified as a 24 promising therapeutic approach to combat multidrug resistant infections. The mechanisms of 25 action, however, remain unclear. A collection strain, Escherichia coli ATCC showing 26 an extended spectrum of resistance to beta-lactam and aminoglycoside antibiotics was used in 27 this study as a model organism. The FS-1 treated culture and the negative control variant were 28 sequenced by PacBio RS II System following the SMRTbell 20-kb library preparation protocol.29 Total RNA samples of these strains were sequenced by Ion Torrent. It was shown that the 30 treatment with FS-1 caused a profound gene expression alternation switching the bacterial 31 metabolism to anaerobic respiration, increased anabolism, oxidative/acidic stress response and an 32 inhibition of many nutrient uptake systems. All this leads to an increase in the susceptibility to 33 antibiotics even when FS-1 is removed from the medium. The later fact implies an involvement 34 of epigenetic mechanisms in gene regulation and antibiotic resistance reversion. This hypothesis 35 was investigated by base-call kinetic analysis in PacBio reads and DNA methylation profiling in 36 the sequenced genomes. Several DNA motifs of adenosine and cytosine methylation were 37 identified. While the numbers of methylated sites in chromosomes and plasmids of both 38 genomes, NC and FS, were similar, the distribution of the methylated sites was different. It may 39 explain the observed long lasting effect of the treatment of E. coli with FS-1 on antibiotic 40 susceptibility of this model microorganism.41 Author summary 42 The emergence of multidrug resistant bacteria is a great concern, since the misuse of antibiotics 43 have caused a strong selective pressure for these resistant bacteria and various treatment options 44 are becoming ineffective. Drug induced reversion of antibiotic resistant is considering a 45 promising approach to address this problem. This study was set out to investigate genetic 46 mechanisms of action of a new iodine-containing nano-micelle drug FS-1 that induces antibiotic 47 resistance reversion in bacteria. Escherichia coli ATCC BAA-196 was used as a model of 48 multidrug resistant microorganisms. With this purpose in mind, we have sequenced the genomic 49 DNA and total RNA samples of E. coli cultivated on medium containing FS-1, served as an 50 experimental (FS) variant, and bacteria cultivated on normal medium, served as negative control 51 (NC). RNA sequencing showed a differential gene expression in the FS-1 treated strain that may 52 explain the observed increase in susceptibility to gentamicin and ampicillin. Application of the 53 3 rd generation sequencing technology, SMRT PacBio, allowed an unambiguous whole genome 54 assembly of NC and FS variants, a...

Chromatographic analysis shows that the ionic nanostructured complex of the FS-1 drug contains nanocomplexes of α-dextrin with a size of ~40–48 Å. Based on good agreement between the UV spectra of the model structures and the experimental spectrum of the FS-1 drug, the structure of the active FS-1 nanocomplex is proposed. The structure of the active centers of the drug in the dextrin ring was calculated using the quantum-chemical approach DFT/B3PW91. The active centers, i.e., a complex of molecular iodine with lithium halide (I), a binuclear complex of magnesium and lithium containing molecular iodine, triiodide (II), and triiodide (III), are located inside the dextrin helix. The polypeptide outside the dextrin helix forms a hydrogen bond with dextrin in Complex I and coordinates the molecular iodine in Complex II. It is revealed that the active centers of the FS-1drug can be segregated from the dextrin helix and form complexes with DNA nucleotide triplets. The active centers of the FS-1 drug are only segregated on specific sections of DNA. The formation of a complex between the DNA nucleotide and the active center of FS-1 is a key stage in the mechanisms of anti-HIV, anti-coronavirus (Complex I) and antibacterial action (Complex II).

Emergence of multidrug resistant strains of Mycobacterium tuberculosis (MDR-TB) threatens humanity. This problem was complicated by the crisis in development of new anti-tuberculosis antibiotics. Induced reversion of drug resistance seems promising to overcome the problem. Successful clinical trial of a new anti-tuberculosis nanomolecular complex FS-1 has demonstrated prospectively of this approach in combating MDR-TB. Several clinical MDR-TB cultures were isolated from sputum samples prior and in the process of the clinical trial. Every isolate was tested for susceptibility to antibiotics and then they were sequenced for comparative genomics. It was found that the treatment with FS-1 caused an increase in the number of antibiotic susceptible strains among Mtb isolates that was associated with a general increase of genetic heterogeneity of the isolates. Observed impairing of phthiocerol dimycocerosate biosynthesis by disruptive mutations in ppsACD subunits indicated a possible virulence remission for the sake of persistence. It was hypothesized that the FS-1 treatment eradicated the most drug resistant Mtb variants from the population by aggravating the fitness cost of drug resistance mutations. Analysis of distribution of these mutations in the global Mtb population revealed that many of them were incompatible with each other and dependent on allelic states of many other polymorphic loci. The latter discovery may explain the negative correlation between the genetic heterogeneity of the population and the level of drug tolerance. To the best of our knowledge, this work was the first experimental confirmation of the drug induced antibiotic resistance reversion by the induced synergy mechanism that previously was predicted theoretically.

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is a leading cause of death in humans worldwide. The emergence of antibiotic-resistant strains of Mtb is a threat to tuberculosis control. A general belief is that drug resistance is acquired by Mtb during antibiotic treatment by accumulation of spontaneous mutations. Also, it is known that the drug resistance mutations (DRM) have an associated fitness cost, reducing the transmissibility and virulence of resistant strains. In this work we show that many canonical DRM are clade specific; i.e. they occur only in specific genetic lineages of Mtb and depend on a specific genetic context necessary for the reduction of the fitness cost and sustainability of the drug resistance phenotype. Dependence of the drug resistance on occurrence of genetic variants of multiple genes and specific activities of the encoded proteins allows combating the drug resistance by impairing the global genetic context. A new drug, FS-1, reverses antibiotic resistance by compromising this genetic context and aggravating the fitness cost of DRM.

Iodine is one of the oldest antimicrobial agents. Until now, there have been no reports on acquiring resistance to iodine. Recent studies showed promising results on application of iodine-containing nano-micelles, FS-1, against antibiotic-resistant pathogens as a supplement to antibiotic therapy. The mechanisms of the action, however, remain unclear. The aim of this study was to perform a holistic analysis and comparison of gene regulation in three phylogenetically distant multidrug-resistant reference strains representing pathogens associated with nosocomial infections from the ATCC culture collection: Escherichia coli BAA-196, Staphylococcus aureus BAA-39, and Acinetobacter baumannii BAA-1790. These cultures were treated by a 5-min exposure to sublethal concentrations of the iodine-containing drug FS-1 applied in the late lagging phase and the middle of the logarithmic growth phase. Complete genome sequences of these strains were obtained in the previous studies. Gene regulation was studied by total RNA extraction and Ion Torrent sequencing followed by mapping the RNA reads against the reference genome sequences and statistical processing of read counts using the DESeq2 algorithm. It was found that the treatment of bacteria with FS-1 profoundly affected the expression of many genes involved in the central metabolic pathways; however, alterations of the gene expression profiles were species specific and depended on the growth phase. Disruption of respiratory electron transfer membrane complexes, increased penetrability of bacterial cell walls, and osmotic and oxidative stresses leading to DNA damage were the major factors influencing the treated bacteria. IMPORTANCE Infections caused by antibiotic-resistant bacteria threaten public health worldwide. Combinatorial therapy in which antibiotics are administered together with supplementary drugs improving susceptibility of pathogens to the regular antibiotics is considered a promising way to overcome this problem. An induction of antibiotic resistance reversion by the iodine-containing nano-micelle drug FS-1 has been reported recently. This drug is currently under clinical trials in Kazakhstan against multidrug-resistant tuberculosis. The effects of released iodine on metabolic and regulatory processes in bacterial cells remain unexplored. The current work provides an insight into gene regulation in the antibiotic-resistant nosocomial reference strains treated with iodine-containing nanoparticles. This study sheds light on unexplored bioactivities of iodine and the mechanisms of its antibacterial effect when applied in sublethal concentrations. This knowledge will aid in the future design of new drugs against antibiotic-resistant infections.