Selection Of Resistant Variants Research Articles

THE SELECTION OF RESISTANT VARIANTS OF OPPORTUNISTIC MICROORGANISMS USING AETHONIUM IN CLINICS FOR 40 YEARS

vitis. Bukovinian Medical Herald. Т.21, № 3 (83). P. 83-90 THE SELECTION OF RESISTANT VARIANTS OF OPPORTUNISTIC MICROORGANISMS USING AETHONIUM IN CLINICS FOR 40 YEARS L.I. Sydorchuk, I.P. Burdeniuk, S.E. Deineka, K.I. Yakovets, I.Y. Sydorchuk, V.I. Burdeniuk1, K.G. Tashchuk Objective: To establish the minimum bacteriostatic and bactericidal concentration of aethonium in relation to conditionally pathogenic microorganisms isolated and identified from the upper respiratory tract of children aged 11-13 years with chronic catarrhal gingivitis with a comorbid state (type I diabetes mellitus) in a comparative sense with sensitivity to aetonium of reference and clinical strains of conditionally pathogenic microorganisms isolated in 1974-1976. Materials and methods. Primary materials of 1974-1976 years of the sensitivity of conditionally pathogenic microorganisms to aethonium were used from dissertational works of co-authors (I.P. Burdeniuk, I.Y. Sydorchuk) for comparison of data obtained in 2014-2016. Microbiological examinations were performed in 30 pupils aged 11-13 years with catarrhal gingivitis with type I diabetes mellitus. The sampling of the material for microbiological examination was carried out with sterile tampons on the oral mucosa, following by seeding the sectors of solid nutrient media optimal for each taxon to obtain pure cultures. To determine the sensitivity of microbes to aethonium, the method of serial two-fold dilutions in a liquid nutrient medium was used. Results. It is shown that the sensitivity of the reference strains of microorganisms for 40 years of storage under laboratory conditions in a semi-solid nutrient medium remains practically unchanged. The bactericidal concentration of aethonium relative to S. faecalis ATCC 29212 increased by 1,96 %. It was shown that the bacteriostatic and bactericidal activity of aethonium decreased over 40 years relative to clinical strains and this decrease (resistance growth) depends on microorganisms, their morphological, tinctorial and other properties. Grampositive and gram-negative clinical strains of microorganisms have decreased sensitivity from 1,21 to 2,56 times. Conclusions. The development of resistant variants of microorganisms proceeds by gradual selection with dissemination in clinics. Before using aethonium, it is necessary to isolate and identify conditionally pathogenic microbes and to establish the bacteriostatic and bactericidal concentration of aethonium with the subsequent solution of the question of its possible effective use.

Second Generation Protease Inhibitors and Nucleotide Inhibitors

The hepatitis C virus (HCV) protease and polymerase inhibitors are in rapid phases of development. Following closely behind the approval of the inhibitors of the HCV RNA NS3/4A protease, boceprevir and telaprevir, include both a) more potent protease inhibitors and b) the development of the viral NS5B RNA-dependent RNA polymerase inhibitors. Protease inhibitors generally have a low barrier to resistance and extensive cross-resistance between agents. NS5B polymerase inhibitors are generally considered to have a high barrier to resistance and the potential for use across all genotypes. However, in the absence of ribavirin or of other direct-acting antivirals these agents may predispose to the selection of resistant variants or for viral relapse following treatment completion. Optimal combination of agents and therapy durations remain major challenges in clinical research.

RG7128 Alone or in Combination with Pegylated Interferon‐α2a and Ribavirin Prevents Hepatitis C Virus (HCV) Replication and Selection of Resistant Variants in HCV‐Infected Patients

RG7128 (prodrug of PSI-6130) shows potent antiviral efficacy in patients infected with hepatitis C virus (HCV) genotypes 1, 2, or 3, with mean viral load decreases of 2.7 and 5 log(10) IU/mL, respectively, associated with 1500-mg doses twice daily after monotherapy for 2 weeks and with 1000-mg and 1500-mg doses twice daily after treatment in combination with the standard of care (SOC) for 4 weeks. From 32 patients treated with RG7128 monotherapy for 2 weeks, marginal viral load rebound was observed in 3 HCV genotype 1-infected patients, whereas partial response was observed in 2 genotype 1-infected patients. From 85 patients receiving RG7128 in combination with SOC, 1 HCV genotype 1-infected patient experienced a viral rebound, and 2 genotype 3-infected patients experienced a transient rebound. Five genotype 1-infected patients had an HCV load of >1000 IU/mL at the end of 4-week treatment. No viral resistance was observed, per NS5B sequencing and phenotypic studies. PSI-6130 resistance substitution S282T needs to be present at levels of ≥90% within a patient's quasispecies to confer low-level resistance. No evidence of S282T was found by population or clonal sequence analyses. The requirement for a predominant S282T mutant quasispecies, its low replication capacity, and the low-level resistance it confers probably contribute to the lack of RG7128 resistance observed in HCV-infected patients.

Resistance mechanisms in HCV: from evolution to intervention

Recent advances in our understanding of the HCV life cycle and the functions of virally encoded proteins enabled the development of specifically targeted antiviral therapies for HCV, which directly inhibit HCV replication. Early clinical trials show great efficacy; however, from the first trials it became evident that, similar to HIV and HBV, selection of resistant variants will be problematic. Error-prone replication of HCV, resulting in a complex quasispecies population within each infected individual, enables rapid adaptation to changing environments. In this review, the evolutionary mechanisms involved in the selection process resulting in drug resistance are discussed. We give an overview of the resistance profiles to recently developed HCV protease and polymerase inhibitors and discuss potential implications for future treatment developments.

Hepatitis B virus genetic diversity

Hepatitis B virus (HBV) is a human DNA virus, which replicates through an RNA intermediate because of the reverse-transcriptase (RT) activity of its DNA polymerase. As a result, the mutation rate for HBV is higher than the rate observed for most DNA viruses. HBVs are classified into genotypes based on genomic sequencing, and antigenic subtypes based on the antigenic properties of its major surface glycoprotein, the HBV surface antigen (HBsAg). Subgenotypes have been identified within most of the HBV genotypes. The HBV groups defined by the different genotype-HBsAg subtype associations found over the world display characteristic geographical distributions, reflecting the movements of human populations and other epidemiologically significant events. Such HBV groups constitute genetically stable viral populations sharing a common evolutionary history, but additional stable changes, originating from mutation and mutant selection, are observed within all of them. These viral sub-populations are known as the HBV variants, and some of which have medical and public health relevance. Pre-core (pre-C) defective variants have been shown to make HBV infection much less susceptible to interferon treatment, and treatment failures with other antiviral drugs have been associated with selection of resistant variants that display specific mutations in the genome region encoding the viral RT activity. Since the RT region of the genome overlaps the sequence encoding the HBsAg molecule, selection of drug resistant variants involves, in some cases, the indirect selection of HBsAg variants. Viral variants displaying changes in HBsAg seem to be very common among chronic HBV carriers; and some of these variants may emerge under the pressure of the neutralizing antibody response, leading to vaccine resistance and resistance to immunotherapy. Mutations conferring resistance to immunotherapy are noted often among liver transplant recipients and among babies born to HBV-carrier mothers. In addition, some of these HBsAg variants have been associated with lack of detection by HBsAg tests used for the diagnosis of HBV infection, for the identification of chronic carriers, for screening of blood donations for transfusion, and in the manufacture of therapeutic blood products.

Intermittent viremia during first-line, protease inhibitors-containing therapy: significance and relationship with drug resistance

In HIV-infected patients on first-line antiretroviral therapy, the significance of intermittent viremia and their relationship with drug resistance remain unclear. To study the virological characteristics of intermittent viremia (IV) and the association between IV and later virological failure (VF) in patients on a first-line, PI-containing therapy. Antiretroviral-naive patients were enrolled in the APROVIR substudy of the prospective, multicenter APROCO cohort at the time they initiated a PI-containing therapy and were followed-up at month 1 and every 2 months. IV was defined as plasma HIV-1 RNA > 500 copies/ml on a single specimen. VF were defined as: (1) viral rebound on two consecutive plasma specimens with HIV-1 RNA > 500 copies/ml after an initial response below 500 copies/ml, or (2) persistence of plasma HIV-1 RNA> or =500 copies/ml during the first year of follow-up. Genotypic resistance analysis was performed at baseline and at the time of IV. PI plasma concentrations were determined at the time of IV. IV was found in 20/219 patients in a 2 years follow-up. The occurrence of IV in the first year of therapy was associated with a higher risk of virological failure during the second year (p = 0.03). Genotypic resistance at the time of IV was found in only 4/16 patients and was not predictive of a subsequent virological failure. PI plasma levels suggested lack of adherence in 50% of patients with IV. The occurrence of IV > 500 copies/ml among patients on first-line, PI-containing ART is suggestive of a lack of adherence rather than the selection of resistant variants and should lead to an intensification of adherence monitoring in order to reduce the risk of subsequent VF.

Resistance to fusidic acid

Resistance to fusidic acid is determined by a number of mechanisms. The best described are alterations in elongation factor G, which appear in natural mutants that are harboured at low rates in normal populations of staphylococci (106 to 108). Altered drug permeability has also been described, and appears to be plasmid-borne. Binding by chloramphenicol acetyltransferase type I and efflux are other described mechanisms of resistance whose prevalence is unclear. A large number of studies have examined rates of fusidic acid resistance in staphylococci. Most show low levels of resistance. Studies where high levels of resistance have been seen are from areas of the hospital where cross infection is common. Rates of resistance have tended to be slightly higher in methicillin-resistant strains of Staphylococcus aureus. Studies on the evolution of resistance have shown no major trends to the emergence of resistance. In one case this is despite increasing use of both systemic and topical fusidic acid over more than 24 years. Selection for resistant variants during treatment was recognised early in vitro and in vivo. However, evidence suggests that it does not occur at high frequency in clinical practice. Nevertheless, accumulated experience is that fusidic acid in combination with other agents results in less resistance emergence.

Modulating the metastatic potential of murine RAW117 large-cell lymphoma cells by selection for resistance to interferon-gamma.

Highly metastatic, in vivo-selected cells of RAW117-H10 large-cell lymphoma have been shown to be more resistant than poorly metastatic parental RAW117-P cells to the cytolytic and cytostatic activities of activated macrophages in co-culture experiments. Activated macrophages are known to produce soluble, cytostatic respiration-inhibiting factors, and such activities can be duplicated by interferon-gamma (IFN-gamma) or by combinations of IFN-gamma and Escherichia coli lipopolysaccharide (LPS). Highly metastatic RAW117-H10 cells are more resistant to the cytostatic effects of IFN-gamma and LPS than poorly metastatic RAW117-P cells, and short-term (up to 72 hr) treatment with IFN-gamma and LPS does not change the metastatic potentials of RAW117 cells. We have studied the effects of sequential selection of RAW117-P cells for increased resistance to IFN-gamma and LPS. After 7 to 13 sequential selections, the resulting variant lines were completely refractory to the growth-inhibitory effects of IFN-gamma and LPS and cross-tolerant to macrophage-conditioned medium. The selected variants also completely lost their experimental metastatic potentials and their tumorigenicities after s.c. or i.m. injection. Cytofluorographic analysis indicated reduced cell-surface expression of H-2Kd antigen and fibronectin receptor on the variant cells but no change in surface mu heavy-chain immunoglobulin. The IFN-gamma-selected lines were less adhesive to liver microvascular endothelial cells than the unselected RAW117 cell lines, but were equally sensitive to NK cytolysis by spleen cells. Our results suggest that exposure to certain cytokines can affect the growth and metastatic potential of RAW117 cells and result in the selection of resistant variants with altered biologic properties.

Frequencies of subpopulations of aminoglycoside- and vancomycin-resistant variants in Staphylococcus aureus and Staphylococcus epidermidis.

Selection and regrowth of resistant variants, which are present in low frequencies in the initial inoculum, were seen when large inocula of five strains of Staphylococcus aureus and four strains of Staphylococcus epidermidis were incubated in broth with amikacin, gentamicin, netilmicin and tobramycin. Statistical analysis showed no significant difference between the aminoglycosides in the selective growth of resistant variants (P > 0.5). Vancomycin differed significantly from the aminoglycosides in both the frequency of, and selection of resistant variants (P < 0.001). No bacteria resistant to > 1 x MIC was seen in the vancomycin-exposed cultures of S. aureus and S. epidermidis, while in most aminoglycoside-exposed cultures, bacteria resistant to 4-16 x MIC were seen.

Antibacterial activity of lomefloxacin in a pharmacokinetic in vitro model

An in vitro model simulating two-compartment pharmacokinetics was used to study the antibacterial activity of lomefloxacin after single oral doses of 200 and 400 mg. Lomefloxacin produced reliable bactericidal activity against gram-negative aerobic bacteria and staphylococci. Bacterial strains for which MICs were less than 0.5 microgram/ml were inhibited with both dosing schedules. Doubling the dose from 200 to 400 mg increased the bactericidal activity only against Pseudomonas aeruginosa. Against Enterococcus faecalis lomefloxacin showed no effect. Selection of resistant variants during the simulation of treatment was observed with Staphylococcus aureus and P. aeruginosa.

Selection of Gentamicin-Resistant Variants of Pseudomonas aeruginosa in the Rat Cornea

Pseudomonas aeruginosa often persists in ocular infections despite intensive antimicrobial therapy. In this study, gentamicin-resistant small colony variants (SCV) were selected in vitro from three gentamicin-susceptible Pseudomonas isolates by incubation with antibiotic. In the rat cornea, SCV were selected from two of these strains by administration of gentamicin either topically or intravenously. The selection of resistant variants may be a contributing factor in the persistence and reactivation frequently associated with ocular Pseudomonas infections.

Inducing capacity and selection of resistant variants of cefpirome (HR 810) in comparison with other beta-lactam compounds.

The inducing capacity of cefpirome (HR 810) and the ability of the compound to select for stable derepressed mutants was determined and compared with those of cefodizime (HR 221), cefotaxime, ceftazidime and cefamandol. Variations in both characteristics between and within species was observed. Overall, cefodizime showed the lowest, cefamandol the highest inducing capacity. Antibiotic resistant variants were isolated from all strains tested at a frequency of around 10(-9). A stable increased enzyme production was found in Pseudomonas aeruginosa after exposure to ceftazidime as well as in the resistant mutants from Enterobacter cloacae after selection with cefpirome, ceftazidime, cefotaxime and cefamandol. In the other resistant mutants the resistance was probably due to changes in permeability. All resistant variants remained relatively susceptible to cefpirome.

Selection frequency of resistant variants by various beta-lactam antibiotics in clinical Enterobacter cloacae isolates.

The frequency of selection of resistant variants by 10 different broad-spectrum beta-lactam derivatives was evaluated for 10 clinical Enterobacter cloacae isolates. With respect to most penams or cephems, resistant variants could be selected up to 8- or 32-fold the MIC, respectively. However, with cefpirome as the selecting agent resistant variants were obtained only at twice the MIC, whereas resistant variants were barely detectable with temocillin and not detectable in any case with imipenem. The variants exhibited cross-resistance between penams and cephems including aztreonam, but not to temocillin and imipenem regardless of the beta-lactamase amount produced. Enzyme production of the variants ranged from 0.2 U to 19.0 U beta-lactamase/mg protein of the cell-free supernatants. Moreover, analysis of the outer membrane protein composition did not reveal marked alterations between wild strains and the corresponding variants. It is evident that 'overproduction of the chromosomal beta-lactamase' cannot explain entirely phenotypic resistance to broad-spectrum beta-lactam compounds, but a lack of porin production, i.e., major outer membrane proteins, cannot provide an explanation for the above findings.

Antibacterial activity of mupirocin (pseudomonic acid), a new antibiotic for topical use.

Mupirocin (pseudomonic acid A), an antibiotic produced by Pseudomonas fluorescens, showed a high level of activity against staphylococci and streptococci and against certain gram-negative bacteria, including Haemophilus influenzae and Neisseria gonorrhoeae, but was much less active against most gram-negative bacilli an anaerobes. Nearly all clinical isolates of Staphylococcus aureus and Staphylococcus epidermidis, including multiply resistant strains, were susceptible (mupirocin MIC, less than or equal to 0.5 microgram/ml). There was no cross-resistance between mupirocin and clinically available antibiotics, and the selection of resistant variants in vitro occurred at a low frequency. Mupirocin was highly bound (95% bound) to the protein of human serum, and activity was reduced 10- to 20-fold in the presence of human serum. The activity of mupirocin was not greatly influenced by inoculum size but was significantly enhanced in acid medium. In tests of bactericidal activity, MBCs were 8- to 32-fold higher than MICs and the antibiotic demonstrated a slow bactericidal action in time-kill tests, resulting in 90 to 99% killing after 24 h at 37 degrees C.

Streptomycin resistance in chloramphenicol-producing strains of Streptomyces species 3022a.

Resistance to low (5 mug/ml) concentrations of streptomycin in agar media was not inherited by all of the surviving population. Outgrowth of cultures in liquid media supplemented with the antibiotic depended upon inoculum size. Antibiotic titers in the supplemented cultures decreased during incubation, and an inactive radioactive product was detected when [14C] streptomycin was used. This low-level resistance is, therefore, attributed to enzymic inactivation of the antibiotic. Growth 10 mug/ml or higher concentrations of streptomycin on agar media was due to selection of resistant variants present in the parent strain. A range of such variants existed, decreasing in frequency as their degree of resistance increased. Examination of one that was resistant to moderate concentrations of streptomycin, (25 mug/ml) and a second that was resistant to high (100 mug/ml) concentrations of streptomycin suggested that both possed ribosomes which had lower affinity for the antibiotic than those of the parent strain, and that tolerance to high levels of streptomycin was due to a resistant ribosomal system for protein biosynthesis.

Action of streptomycin on the growth of Streptomyces griseus.

A streptomycin-producing culture of Streptomyces griseus was sensitive to streptomycin, but inhibition was temporary, and cultures supplemented with the antibiotic up to 100 μg/ml grew after a lag. Streptomycin tolerance developed, not by inactivation of the antibiotic but by selection of resistant variants in the natural population. Addition of streptomycin to growing cultures caused a drastic reduction in protein synthesis and an accumulation of "stuck" (70 S) ribosomes within the mycelium. Although the effect on protein synthesis could not be confirmed in vitro because cell extracts from S. griseus contained an inhibitor of polyuridylic-acid-directed polymerization of L-phenylalanine, it is concluded that streptomycin prevents the growth of this organism by a mechanism similar to that observed with other bacteria and that the tolerance of producing cultures cannot be attributed to the presence of streptomycin-resistant ribosomes.