Antibiotic resistance begets more resistance: chromosomal resistance mutations mitigate fitness costs conferred by multi-resistant clinical plasmids.

Plasmids are the primary vectors of horizontal transfer of antibiotic resistance genes among bacteria. Previous studies have shown that the spread and maintenance of plasmids among bacterial populations depend on the genetic makeup of both the plasmid and the host bacterium. Antibiotic resistance can also be acquired through mutations in the bacterial chromosome, which not only confer resistance but also result in changes in bacterial physiology and typically a reduction in fitness. However, it is unclear whether chromosomal resistance mutations affect the interaction between plasmids and the host bacteria. To address this question, we introduced 13 clinical plasmids into a susceptible Escherichia coli strain and three different congenic mutants that were resistant to nitrofurantoin (ΔnfsAB), ciprofloxacin (gyrA, S83L), and streptomycin (rpsL, K42N) and determined how the plasmids affected the exponential growth rates of the host in glucose minimal media. We find that though plasmids confer costs on the susceptible strains, those costs are fully mitigated in the three resistant mutants. In several cases, this results in a competitive advantage of the resistant strains over the susceptible strain when both carry the same plasmid and are grown in the absence of antibiotics. Our results suggest that bacteria carrying chromosomal mutations for antibiotic resistance could be a better reservoir for resistance plasmids, thereby driving the evolution of multi-drug resistance.IMPORTANCEPlasmids have led to the rampant spread of antibiotic resistance genes globally. Plasmids often carry antibiotic resistance genes and other genes needed for its maintenance and spread, which typically confer a fitness cost on the host cell observed as a reduced growth rate. Resistance is also acquired via chromosomal mutations, and similar to plasmids they also reduce bacterial fitness. However, we do not know whether resistance mutations affect the bacterial ability to carry plasmids. Here, we introduced 13 multi-resistant clinical plasmids into a susceptible and three different resistant E. coli strains and found that most of these plasmids do confer fitness cost on susceptible cells, but these costs disappear in the resistant strains which often lead to fitness advantage for the resistant strains in the absence of antibiotic selection. Our results imply that already resistant bacteria are a more favorable reservoir for multi-resistant plasmids, promoting the ascendance of multi-resistant bacteria.

Plasmid fitness costs are caused by specific genetic conflicts enabling resolution by compensatory mutation.

Plasmids play an important role in bacterial genome evolution by transferring genes between lineages. Fitness costs associated with plasmid carriage are expected to be a barrier to gene exchange, but the causes of plasmid fitness costs are poorly understood. Single compensatory mutations are often sufficient to completely ameliorate plasmid fitness costs, suggesting that such costs are caused by specific genetic conflicts rather than generic properties of plasmids, such as their size, metabolic burden, or gene expression level. By combining the results of experimental evolution with genetics and transcriptomics, we show here that fitness costs of 2 divergent large plasmids in Pseudomonas fluorescens are caused by inducing maladaptive expression of a chromosomal tailocin toxin operon. Mutations in single genes unrelated to the toxin operon, and located on either the chromosome or the plasmid, ameliorated the disruption associated with plasmid carriage. We identify one of these compensatory loci, the chromosomal gene PFLU4242, as the key mediator of the fitness costs of both plasmids, with the other compensatory loci either reducing expression of this gene or mitigating its deleterious effects by up-regulating a putative plasmid-borne ParAB operon. The chromosomal mobile genetic element Tn6291, which uses plasmids for transmission, remained up-regulated even in compensated strains, suggesting that mobile genetic elements communicate through pathways independent of general physiological disruption. Plasmid fitness costs caused by specific genetic conflicts are unlikely to act as a long-term barrier to horizontal gene transfer (HGT) due to their propensity for amelioration by single compensatory mutations, helping to explain why plasmids are so common in bacterial genomes.

Enhanced copper-resistance gene repertoire in Alteromonas macleodii strains isolated from copper-treated marine coatings.

Copper is prevalent in coastal ecosystems due to its use as an algaecide and as an anti-fouling agent on ship hulls. Alteromonas spp. have previously been shown to be some of the early colonizers of copper-based anti-fouling paint but little is known about the mechanisms they use to overcome this initial copper challenge. The main models of copper resistance include the Escherichia coli chromosome-based Cue and Cus systems; the plasmid-based E. coli Pco system; and the plasmid-based Pseudomonas syringae Cop system. These were all elucidated from strains isolated from copper-rich environments of agricultural and/or enteric origin. In this work, copper resistance assays demonstrated the ability of Alteromonas macleodii strains CUKW and KCC02 to grow at levels lethal to other marine bacterial species. A custom database of Hidden Markov Models was designed based on proteins from the Cue, Cus, and Cop/Pco systems and used to identify potential copper resistance genes in CUKW and KCC02. Comparative genomic analyses with marine bacterial species and bacterial species isolated from copper-rich environments demonstrated that CUKW and KCC02 possess genetic elements of all systems, oftentimes with multiple copies, distributed throughout the chromosome and mega-plasmids. In particular, two copies of copA (the key player in cytoplasmic detoxification), each with its own apparent MerR-like transcriptional regulator, occur on a mega-plasmid, along with multiple copies of Pco homologs. Genes from both systems were induced upon exposure to elevated copper levels (100 µM- 3 mM). Genomic analysis identified one of the merR-copA clusters occurs on a genomic island (GI) within the plasmid, and comparative genomic analysis found that either of the merR-copA clusters, which also includes genes coding for a cupredoxin domain-containing protein and an isoprenylcysteine methyltransferase, occurs on a GI across diverse bacterial species. These genomic findings combined with the ability of CUKW and KCC02 to grow in copper-challenged conditions are couched within the context of the genome flexibility of the Alteromonas genus.

In vitro mutagenicity assessment of fried meat-based food from mass catering companies.

The current article aimed to evaluate the in vitro mutagenicity of ten fried meat-based food extracts obtained from different catering companies from Navarra (Spain). A miniaturized 6-well version of the Ames test in Salmonella typhimurium TA98, and the in vitro micronucleus test (OECD TG 487) in TK6 cells were performed. None of the ten extracts of fried meat-based food induced gene mutations in S. typhimurium TA98 with or without metabolic activation, but five induced chromosomal aberrations after 24 h treatment of TK6 without metabolic activation. More studies are needed to check the biological relevance of these in vitro studies.

Engineered Recombinant Escherichia coli Probiotic Strains Integrated with F4 and F18 Fimbriae Cluster Genes in the Chromosome and Their Assessment of Immunogenic Efficacy in Vivo.

F4 (K88) and F18 fimbriaed enterotoxigenic Escherichia coli (ETEC) are the predominant causes of porcine postweaning diarrhea (PWD), and vaccines are considered the most effective preventive approach against PWD. Since heterologous DNA integrated into bacterial chromosomes could be effectively expressed with stable inheritance, we chose probiotic EcNc (E. coli Nissle 1917 prototype cured of cryptic plasmids) as a delivery vector to express the heterologous F4 or both F4 and F18 fimbriae and sequentially assessed their immune efficacy of anti-F4 and F18 fimbriae in both murine and piglet models. Employing the CRISPR-cas9 technology, yjcS, pcadA, lacZ, yieN/trkD, maeB, and nth/tppB sites in the chromosome of an EcNc strain were targeted as integration sites to integrate F4 or F18 fimbriae cluster genes under the Ptet promotor to construct two recombinant integration probiotic strains (RIPSs), i.e., nth integration strain (EcNcΔnth/tppB::PtetF4) and multiple integration strain (EcNc::PtetF18x4::PtetF4x2). Expression of F4, both F4 and F18 fimbriae on the surfaces of two RIPSs, was verified with combined methods of agglutination assay, Western blot, and immunofluorescence microscopy. The recombinant strains have improved adherence to porcine intestinal epithelial cell lines. Mice and piglets immunized with the nth integration strain and multiple integration strain through gavage developed anti-F4 and both anti-F4 and anti-F18 IgG immune responses. Moreover, the serum antibodies from the immunized mice and piglets significantly inhibited the adherence of F4+ or both F4+ and F18+ ETEC wild-type strains to porcine intestinal cell lines in vitro, indicating the potential of RIPSs as promising probiotic strains plus vaccine candidates against F4+/F18+ ETEC infection.

A lattice kinetic Monte-Carlo method for simulating chromosomal dynamics and other (non-)equilibrium bio-assemblies.

Biological assemblies in living cells such as chromosomes constitute large many-body systems that operate in a fluctuating, out-of-equilibrium environment. Since a brute-force simulation of that many degrees of freedom is currently computationally unfeasible, it is necessary to perform coarse-grained stochastic simulations. Here, we develop all tools necessary to write a lattice kinetic Monte-Carlo (LKMC) algorithm capable of performing such simulations. We discuss the validity and limits of this approach by testing the results of the simulation method in simple settings. Importantly, we illustrate how at large external forces Metropolis-Hastings kinetics violate the fluctuation-dissipation and steady-state fluctuation theorems and discuss better alternatives. Although this simulation framework is rather general, we demonstrate our approach using a DNA polymer with interacting SMC condensin loop-extruding enzymes. Specifically, we show that the scaling behavior of the loop-size distributions that we obtain in our LKMC simulations of this SMC-DNA system is consistent with that reported in other studies using Brownian dynamics simulations and analytic approaches. Moreover, we find that the irreversible dynamics of these enzymes under certain conditions result in frozen, sterically jammed polymer configurations, highlighting a potential pitfall of this approach.

Bacterial chromosome organization. I. Crucial role of release of topological constraints and molecular crowders.

We showed in our previous studies that just 3% cross-links (CLs), at special points along the contour of the bacterial DNA, help the DNA-polymer to get organized at micron length scales [T. Agarwal et al., J. Phys.: Condens. Matter 30, 034003 (2018) and T. Agarwal et al., EPL (Europhys. Lett.) 121, 18004 (2018)]. In this work, we investigate how does the release of topological constraints help in the "organization" of the DNA-polymer. Furthermore, we show that the chain compaction induced by the crowded environment in the bacterial cytoplasm contributes to the organization of the DNA-polymer. We model the DNA chain as a flexible bead-spring ring polymer, where each bead represents 1000 base pairs. The specific positions of the CLs have been taken from the experimental contact maps of the bacteria Caulobacter crescentus and Escherichia coli. We introduce different extents of ease of release of topological constraints in our model by systematically changing the diameter of the monomer bead. It varies from the value where chain crossing can occur freely to the value where chain crossing is disallowed. We also study the role of compaction of the chain due to molecular crowders by introducing an "effective" weak Lennard-Jones attraction between the monomers. Using Monte Carlo simulations, we show that the release of topological constraints and the crowding environment play a crucial role to obtain a unique organization of the polymer.

Bacterial chromosome organization. II. Few special cross-links, cell confinement, and molecular crowders play the pivotal roles.

Using a coarse-grained bead-spring model of bacterial chromosomes of Caulobacter crescentus and Escherichia coli, we show that just 33 and 38 effective cross-links in 4017 and 4642 monomer chains at special positions along the chain contour can lead to the large-scale organization of the DNA polymer, where confinement effects of the cell walls play a key role in the organization. The positions of the 33/38 cross-links along the chain contour are chosen from the Hi-C contact map of bacteria C. crescentus and E. coli. We represent 1000 base pairs as a coarse-grained monomer in our bead-spring flexible ring polymer model of the DNA polymer. Thus, 4017/4642 beads on a flexible ring polymer represent the C. crescentus/E. coli DNA polymer with 4017/4642 kilo-base pairs. Choosing suitable parameters from Paper I, we also incorporate the role of compaction of the polymer coil due to the presence of molecular crowders and the ability of the chain to release topological constraints. We validate our prediction of the organization of the bacterial chromosomes with available experimental data and also give a prediction of the approximate positions of different segments within the cell. In the absence of confinement, the minimal number of effective cross-links required to organize the DNA chains of 4017/4642 monomers was 60/82 [Agarwal et al., Europhys. Lett. 121, 18004 (2018) and Agarwal et al., J. Phys.: Condens. Matter 30, 034003 (2018)].

Reduction of the fitness cost of antibiotic resistance caused by chromosomal mutations under poor nutrient conditions.

The prevalence of antibiotic resistance in drinking water system is pressing public health risk. Antibiotic resistance conferred by chromosomal mutations often produces fitness cost, which may affect its spread and persistence. In this study, the rifampin-resistant strains were competed with their wild-type counterparts at different nutrient levels. It was observed that the ratio of the absolute number between resistant and wild-type cells quickly decreased under rich nutrient conditions, but it slowly reduced or remained stable in the poor nutrient medium. This finding suggested that poor nutrient conditions resulted in the reduction of fitness cost of antibiotic resistance, i.e. the resistant bacteria became more competitive. Implying mechanisms analysis found that the differences of metabolic activity between wild-type and rifampin-resistant strains was significant smaller (P < 0.05) at low nutrient levels. Additionally, distinguishable large colony size rifampin-resistant strains were observed during competition assay. DNA sequencing of RNA polymerase subunit genes further revealed that these colonies could be adaptive mutants from wild-type strain in rpoB gene. To our knowledge, this is the first study to reveal that the oligotrophic conditions facilitate the persistence of antibiotic resistance in drinking water by reducing the fitness cost of the resistant strains.

Assessment of Chromosomal DNA Fragmentation by Quinolones in an Isogenic Collection of Escherichia coli with Defined Resistance Mechanisms.

The aim of this study was to investigate the potential usefulness of DNA fragmentation as a quick and simple procedure for detecting resistance to fluoroquinolones (FQ) in isogenic Escherichia coli strains harboring defined and multiple quinolone resistance mechanisms, including low-level quinolone resistance (LLQR) phenotypes. DNA fragmentation assay (Micromax(®)) was evaluated for detecting resistance to FQ in 71 isogenic strains of E. coli harboring specific quinolone resistance mechanisms frequently found in clinical isolates. These isogenic strains represent a consistent and reliable model of increasing minimum inhibitory concentrations (MICs) of ciprofloxacin (CIP), ranging from 0.004 to 16 mg/L. According to CLSI criteria, the assay correctly identified all CIP-resistant strains (MIC ≥4 mg/L). As regards susceptible strains, 96% of bacterial strains were correctly assigned as susceptible to CIP. Moreover, the procedure enabled LLQR phenotypes to be efficiently identified; this subset may show different levels of DNA damage depending on the strain, even with similar MIC. Interestingly, despite increasing the dose according to the MIC, a lower response to quinolones occurs in strains with higher MIC values. This is a simple, rapid, and reliable test for evaluating susceptibility to FQ of E. coli, including the detection of strains harboring LLQR mechanisms.

Identifying essential genes in Mycobacterium tuberculosis by global phenotypic profiling.

Transposon sequencing (TnSeq) is a next-generation deep sequencing-based method to quantitatively assess the composition of complex mutant transposon libraries after pressure from selection. Although this method can be used for any organism in which transposon mutagenesis is possible, this chapter describes its use in Mycobacterium tuberculosis. More specifically, the methods for generating complex libraries through transposon mutagenesis, design of selective pressure, extraction of genomic DNA, amplification and quantification of transposon insertions through next-generation deep sequencing are covered. Determining gene essentiality and statistical analysis on data collected are also discussed.

Markovianness and conditional independence in annotated bacterial DNA.

We explore the probabilistic structure of DNA in a number of bacterial genomes and conclude that a form of Markovianness is present at the boundaries between coding and non-coding regions, that is, the sequence of START and STOP codons annotated for the bacterial genome. This sequence is shown to satisfy a conditional independence property which allows its governing Markov chain to be uniquely identified from the abundances of START and STOP codons. Furthermore, we show that the annotated sequence of STARTs and STOPs complies with Chargaff's second parity rule.

Condensation and localization of the partitioning protein ParB on the bacterial chromosome.

The ParABS system mediates chromosome segregation and plasmid partitioning in many bacteria. As part of the partitioning mechanism, ParB proteins form a nucleoprotein complex at parS sites. The biophysical basis underlying ParB-DNA complex formation and localization remains elusive. Specifically, it is unclear whether ParB spreads in 1D along DNA or assembles into a 3D protein-DNA complex. We show that a combination of 1D spreading bonds and a single 3D bridging bond between ParB proteins constitutes a minimal model for a condensed ParB-DNA complex. This model implies a scaling behavior for ParB-mediated silencing of parS-flanking genes, which we confirm to be satisfied by experimental data from P1 plasmids. Furthermore, this model is consistent with experiments on the effects of DNA roadblocks on ParB localization. Finally, we show experimentally that a single parS site is necessary and sufficient for ParB-DNA complex formation in vivo. Together with our model, this suggests that ParB binding to parS triggers a conformational switch in ParB that overcomes a nucleation barrier. Conceptually, the combination of spreading and bridging bonds in our model provides a surface tension ensuring the condensation of the ParB-DNA complex, with analogies to liquid-like compartments such as nucleoli in eukaryotes.

Modelling of crowded polymers elucidate effects of double-strand breaks in topological domains of bacterial chromosomes.

Using numerical simulations of pairs of long polymeric chains confined in microscopic cylinders, we investigate consequences of double-strand DNA breaks occurring in independent topological domains, such as these constituting bacterial chromosomes. Our simulations show a transition between segregated and mixed state upon linearization of one of the modelled topological domains. Our results explain how chromosomal organization into topological domains can fulfil two opposite conditions: (i) effectively repulse various loops from each other thus promoting chromosome separation and (ii) permit local DNA intermingling when one or more loops are broken and need to be repaired in a process that requires homology search between broken ends and their homologous sequences in closely positioned sister chromatid.

Safety assessment of mushroom ß-glucan: subchronic toxicity in rodents and mutagenicity studies.

Mushroom ß-glucan, a polymer of ß-(1,3/1,6)-glucan, has been claimed for its health benefits. The objective of this study was to assess the safety in-use of mushroom ß-glucan as dietary supplement and food ingredient. Hence, a subchronic toxicity and mutagenicity studies were conducted. In the subchronic toxicity study, Sprague Dawley rats (12/sex/group) were administered (gavage) mushroom ß-glucan at dose levels of 0, 500, 1000 and 2000 mg/kg body weight (bw)/day for 90 days. As compared to control group, administration of ß-glucan did not result in any toxicologically significant treatment-related changes in clinical observations, ophthalmic examinations, body weights, body weight gains, feed consumption, and organ weights. No adverse effects of the ß-glucan on the hematology, serum chemistry parameters, urinalysis or terminal necropsy (gross or histopathology findings) were noted. The results of mutagenicity studies as evaluated by gene mutations in Salmonella typhimurium, in vitro chromosome aberrations and in vivo micronucleus test in mouse did not reveal any genotoxicity of ß-glucan. Based on the subchronic study, the no observed-adverse-effect level (NOAEL) for mushroom ß-glucan was determined as 2000 mg/kgbw/day, the highest dose tested.

MRSA SCCmec epidemiology in Israel: development and implementation of an MRSA SCCmec typing strategy.

Staphylococcal cassette chromosome mec (SCCmec) typing is essential for investigating the molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA). Published assays were often found to be limited in their ability to characterize isolates that were not collected locally. We aimed to develop, test, and implement a molecular method which would enable the SCCmec classification of a large collection of MRSA isolates in a routine clinical laboratory. A multistep working algorithm consisting of two main steps and four additional supplementary steps was developed, using previously reported primers. A total of 1,008 isolates obtained locally, by both clinical and screening cultures, were tested. The majority of isolates (82.54%) could be classified using two main reactions. Overall, our MRSA SCCmec typing strategy was able to classify 917/1,008 (90.97%) of the MRSA isolates. The most predominant was type II SCCmec (41%); a high prevalence of type V SCCmec (16.37%) was also found. PVL gene carriage was found among two type IV SCCmec isolates only. We present a logistically feasible, multistep, MRSA SCCmec typing algorithm which can be used to type a large collection of MRSA isolates. The distinctly higher prevalence of SCCmec type V might reflect a unique MRSA distribution pattern in Israel.

A geometrical model for DNA organization in bacteria.

Recent experimental studies have revealed that bacteria, such as C. crescentus, show a remarkable spatial ordering of their chromosome. A strong linear correlation has been found between the position of genes on the chromosomal map and their spatial position in the cellular volume. We show that this correlation can be explained by a purely geometrical model. Namely, self-avoidance of DNA, specific positioning of one or few DNA loci (such as origin or terminus) together with the action of DNA compaction proteins (that organize the chromosome into topological domains) are sufficient to get a linear arrangement of the chromosome along the cell axis. We develop a Monte-Carlo method that allows us to test our model numerically and to analyze the dependence of the spatial ordering on various physiologically relevant parameters. We show that the proposed geometrical ordering mechanism is robust and universal (i.e. does not depend on specific bacterial details). The geometrical mechanism should work in all bacteria that have compacted chromosomes with spatially fixed regions. We use our model to make specific and experimentally testable predictions about the spatial arrangement of the chromosome in mutants of C. crescentus and the growth-stage dependent ordering in E. coli.

Development of a microfluidic chip-based plasmid miniprep.

Plasmids are the workhorse of contemporary molecular biology, serving as vectors in the multitude of molecular cloning approaches now available. Plasmid minipreps are a routine and essential means of extracting plasmid DNA from bacteria, such as Escherichia coli, for identification, characterization, and further manipulation. Although there have been many approaches described and miniprep kits are commercially available, traditional minipreps typically require more than 16h, including the time needed for bacterial cell culture. Here we describe the development of a microfluidic chip (MFC)-based miniprep that uses on-chip lysis and trapping of large DNA in agarose to differentially separate plasmid DNA from the bacterial chromosome. Our approach greatly decreases both the time required for the miniprep itself and the time required for growth of the bacterial cultures because our on-chip miniprep uses 10(5) times fewer E. coli cells. Because the quality of the isolated plasmid is comparable to that obtained using conventional miniprep protocols, this approach allows growth of E. coli and isolation of plasmid within hours, thereby making it ideal for rapid screening approaches. This MFC-based miniprep, coupled with recently demonstrated on-chip transfection capabilities, lays the groundwork for seamless manipulation of plasmids on MFC platforms.

Multiplex real-time PCR for rapid Staphylococcal cassette chromosome mec typing.

Rapid identification and typing of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is important for understanding the molecular epidemiology and evolution of MRSA and offers many advantages for controlling transmission in both health care and community settings. We developed a rapid molecular beacon real-time PCR (MB-PCR) assay for staphylococcal cassette chromosome mec (SCCmec) typing. The design of this system is based on the established definition of SCCmec types, namely, the combination of the mec class complex with the ccr allotype. The assay consists of two multiplex panels, the combination of which results in two targets (mec class, ccr) for each SCCmec type. MB-PCR panel I targets mecA, ccrB2, mecI, and the DeltamecR1-IS1272 junction (mec class B); it can definitively identify SCCmec types II and IV. MB-PCR panel II detects ccrC, ccrB1, ccrB3, ccrB4, and the DeltamecR1-IS431 junction (mec class C2) and is therefore capable of identifying SCCmec types I, III, V, and VI in combination with panel I. The method can also detect the recently described novel SCCmec type VIII (ccrAB4 with mec class A). Our assay demonstrated 100% concordance when applied to 162 MRSA strains previously characterized by traditional SCCmec typing schemes. Four geographically and temporally diverse S. aureus collections were also successfully classified by our assay, along with 1,683 clinical isolates comprising both hospital- and community-associated MRSA and methicillin-susceptible S. aureus strains. As many as 96 isolates can be classified easily within 3 to 4 h, including DNA isolation, PCR cycling, and analysis. The assay is rapid, robust, sensitive, and cost-effective, allowing for high-throughput SCCmec typing of MRSA isolates.

The in vitro fitness cost of antimicrobial resistance in Escherichia coli varies with the growth conditions.

The objective of this study was to investigate the influence of stressful growth conditions on the fitness cost of antimicrobial resistance in Escherichia coli BJ4 caused by chromosomal mutations and plasmid acquisition. The fitness cost of chromosomal streptomycin resistance increased significantly when the bacteria were grown under all stress conditions tested, while the cost in 1/3 Luria-Bertani was not significantly changed in a streptomycin+rifampicin mutant. The increase in the fitness cost depended in a nonregular manner on the strain/stress combination. The fitness cost of plasmid-encoded resistance on R751 did not differ significantly, and was generally less under stressful growth conditions than in rich media. The fitness cost associated with R751 with the multiple drug resistance cassette from Salmonella Typhimurium DT104 increased significantly only under stressful conditions at low pH and at high-salt concentrations. Strains with an impaired rpoS demonstrated a reduced fitness only during growth in a high-salt concentration. In conclusion, it was demonstrated that bacterial fitness cost in association with antimicrobial resistance generally increases under stressful growth conditions. However, the growth potential of bacteria with antimicrobial resistances did not increase in a straightforward manner in these in vitro experiments and is therefore probably even more difficult to predict in vivo.