NERAZLIKOVNOST RAZLIKA

Analiziraju se leksikološko-leksikografska polazišta i metodološki postupci u izradi Razlikovnog rječnika srpskog i hrvatskog jezika Vladimira Brodnjaka

A phase-variable Type I restriction modification system as a bacteriophage defence mechanism and virulence regulator

Streptococcus pneumoniae is one of the most dangerous threats to global health, being the leading cause of acute invasive infection including community acquired pneumonia, meningitis and acute otitis media. The switching between two phenotypic phases, the transparent and opaque colony morphology phenotypes is associated to asymptomatic carriage or invasive disease. Despite being described long time ago, no molecular mechanisms underlying such phase variation were identified. Genetic rearrangements in a pneumococcal Type I restriction methylation (R-M) system locus were previously noticed at the time of the whole genome sequencing of the S. pneumoniae invasive isolate TIGR4 and similarly described in Mycoplasma pulmonis and Bacteroides fragilis. In the pneumococcus this R-M system has been named SpnD39III. In particular, all the six possible rearrangements between multiple hsdS genes were described and the different target specificities identified. Single-molecule, real-time (SMRT) methylomics allowed for the association of the distinct methylation patterns to each SpnD39III conformation. SpnD39III locked mutants allowed the identification of distinct gene expression profiles for each enzyme variant. Consistently with this finding, the SpnD39III system was shown to be involved in the transparent and opaque phenotypes. More importantly, experimental animal models of infection showed variable virulence levels related to each hsdS conformation and in vivo selection for switching between SpnD39III variants. In summary, given the abovementioned results, it has been proposed that the phase-variable SpnD39III R-M system epigenetically regulates gene expression and virulence. This part of the thesis, which is reported in chapter II, has been recently published on Nature Communications. In order to identify the relative amount of each SpnD39III conformation in a pneumococcal population, a quantitative method has been developed and optimised as shown in chapter III. The protocol consists of a first common PCR using a fluorescent-labelled primer, a differentiating restriction digestion, and a final quantitative GeneScan analysis. This method was shown to be sensitive and reproducible. In chapter IV, interactions between the SpSL1 temperate bacteriophage and its pneumococcal host were investigated in order to better understand their co-evolutionary dynamics. The infection kinetics of the phage as well as the integration in the pneumococcal genome were described. The lytic phage infection showed an impact on global host transcriptome, with the majority of changes, occurring transiently in the early stage of infection. The observed variations were typical of a metabolic stress related response. In addition, the SpnIII R-M system was shown to be effective in restricting invading phage proportionally to the amount of target sites in the phage genome. Phase-variation of the SpnIII R-M system was also shown to limit intra-strain propagation of phage. Interestingly, despite restriction, SpSL1 infected cells showed the induction of cell death. Deletion of the pneumococcal McrBC Type IV R-M system, specific for methylated cytosines, decreased significantly the abortive infection mechanism, thereby indicating that chromosomal methylation by the phage-encoded C5-cytosine methyltransferase might be the cause of induced cell death. These data demonstrate the function of McrBC system as a novel phage abortive infection mechanism that is triggered by phage methylation of the bacterial chromosome. The appendix section, at the end of the thesis, contains the result of side projects carried out during the PhD that are beyond the aims of this thesis. In summary the work presented in this thesis describes the discovery of an intricate interplay of methylation systems between the bacterial host and a virus. The phase variable Type I R-M system SpnIII of pneumococci shows both epigenetic control over host phenotypes and inter and intra-strain restriction of phage infection. On the other hand a phage-encoded methylase is involved in a novel abortive infection mechanism by methylating host DNA and determining cell death via the host-encoded SpnMcrBC type IV R-M system. Such interplay between methylases and restriction modules sheds new light on the dynamics of host-phage interaction

Recent advances in the potential interconnection between antimicrobial resistance to biocides and antibiotics

none5siInterconnection between microbial resistance to biocides and antibiotics is a topic of increasing interest given the recent changes in European legislation and claims of a risk of biocide use on bacterial resistance. In the second International Conference on Antimicrobial Research held in Lisbon in November 2012, a workshop specifically addressed this topic, presentations included approaches to risk assessment and investigations into the molecular mechanisms of biocide resistance and co- and cross-resistance to antibiotics. The overall conclusion was that, even if each biocide represents a specific case, there is scientific evidence that biocides select for biocide resistance, but that there is, so far, no conclusive evidence that this also determined or will determine an increase in antibiotic resistance.mixedOggioni MR; L. Furi; JR Coelho; JY Maillard; JL MartinezOggioni MR; L. Furi; JR Coelho; JY Maillard; JL Martine

Is adsorption an artifact in experimentation with Triclosan?

This paper examines the effect of adsorption of Triclosan (TCS) onto labware on the results obtained during lab-scale experiments. Three sets of experiments were considered; two of them expose the problem in water or wastewater treatability studies and the other one in microbial susceptibility testings. In the former two sets, lab-scale systems; ozonation; and membrane filtration (NF/RO) that are commonly used in water or wastewater treatability studies were utilized and the distribution of TCS within the systems were followed. The ozonation labware tested was composed of a Pyrex reactor with plastic and glass tubings. The NF/RO system was composed of a stainless steel feed tank, a stainless steel membrane unit, stainless steel flanges, and stainless steel and plastic tubings. Ozonation system was operated without ozone gas, but air. Similarly, NF/RO system was without membrane in it. Both of the systems were rinsed with methanol before experiments to remove any possible earlier contamination. During the experiments, samples were taken at certain intervals and the change in TCS concentration in water was monitored. Results obtained with lab-scale ozonation system revealed that TCS adsorbed by the surface of plastic tubing is about 100 times greater than that of glass tubing. In NF/RO system, the higher the initial TCS concentration the higher the mass of TCS adsorbed by the membrane filtration system alone was evident. In the third set, microbial susceptibility testing was conducted on Staphylococcus aureus for TCS and the possible effect of adsorption of TCS onto the plastic labware was sought by comparing MIC and MBC values performed by serial dilutions in aqueous and methanol solutions. MIC and MBC values determined using TCS in methanol range from 0.06 to 16 mg/L, while the ones determined with TCS in water range from 0.25 to 128 mg/L. All the results obtained indicated that adsorption is a substantial phenomenon; in the event that it is not considered, obtained results might not reflect the truth. TCS was found to adsorb seriously on plastic but not on glass labwares. Therefore, before an experimental system that will employ TCS is designed, it is essential to consider the possible adsorption onto the experimental system components and to demonstrate that there is no adsorption of TCS onto labware

Mutations upstream of fabI in triclosan resistant Staphylococcus aureus strains are associated with elevated fabI gene expression

Background The enoyl-acyl carrier protein (ACP) reductase enzyme (FabI) is the target for a series of antimicrobial agents including novel compounds in clinical trial and the biocide triclosan. Mutations in fabI and heterodiploidy for fabI have been shown to confer resistance in S. aureus strains in a previous study. Here we further determined the fabI upstream sequence of a selection of these strains and the gene expression levels in strains with promoter region mutations. Results Mutations in the fabI promoter were found in 18% of triclosan resistant clinical isolates, regardless the previously identified molecular mechanism conferring resistance. Although not significant, a higher rate of promoter mutations were found in strains without previously described mechanisms of resistance. Some of the mutations identified in the clinical isolates were also detected in a series of laboratory mutants. Microarray analysis of selected laboratory mutants with fabI promoter region mutations, grown in the absence of triclosan, revealed increased fabI expression in three out of four tested strains. In two of these strains, only few genes other than fabI were upregulated. Consistently with these data, whole genome sequencing of in vitro selected mutants identified only few mutations except the upstream and coding regions of fabI, with the promoter mutation as the most probable cause of fabI overexpression. Importantly the gene expression profiling of clinical isolates containing similar mutations in the fabI promoter also showed, when compared to unrelated non-mutated isolates, a significant up-regulation of fabI. Conclusions In conclusion, we have demonstrated the presence of C34T, T109G, and A101C mutations in the fabI promoter region of strains with fabI up-regulation, both in clinical isolates and/or laboratory mutants. These data provide further observations linking mutations upstream fabI with up-regulated expression of the fabI gene

Significant Differences Characterise the Correlation Coefficients between Biocide and Antibiotic Susceptibility Profiles in Staphylococcus aureus

There is a growing concern by regulatory authorities for the selection of antibiotic resistance caused by the use of biocidal products. We aimed to complete the detailed information on large surveys by investigating the relationship between biocide and antibiotic susceptibility profiles of a large number of Staphylococcus aureus isolates using four biocides and antibiotics commonly used in clinical practice. The minimal inhibitory concentration (MIC) for most clinically-relevant antibiotics was determined according to the standardized methodology for over 1600 clinical S. aureus isolates and compared to susceptibility profiles of benzalkonium chloride, chlorhexidine, triclosan, and sodium hypochlorite. The relationship between antibiotic and biocide susceptibility profiles was evaluated using non-linear correlations. The main outcome evidenced was an absence of any strong or moderate statistically significant correlation when susceptibilities of either triclosan or sodium hypochlorite were compared for any of the tested antibiotics. On the other hand, correlation coefficients for MICs of benzalkonium chloride and chlorhexidine were calculated above 0.4 for susceptibility to quinolones, beta-lactams, and also macrolides. Our data do not support any selective pressure for association between biocides and antibiotics resistance and furthermore do not allow for a defined risk evaluation for some of the compounds. Importantly, our data clearly indicate that there does not involve any risk of selection for antibiotic resistance for the compounds triclosan and sodium hypochlorite. These data hence infer that biocide selection for antibiotic resistance has had so far a less significant impact than feared

Dissemination of Novel Antimicrobial Resistance Mechanisms through the Insertion Sequence Mediated Spread of Metabolic Genes.

The widely used biocide triclosan selectively targets FabI, the NADH-dependent trans-2-enoyl-acyl carrier protein (ACP) reductase, which is also an important target for the development of narrow spectrum antibiotics. The analysis of triclosan resistant Staphylococcus aureus isolates had previously shown that in about half of the strains, the mechanism of triclosan resistance consists on the heterologous duplication of the triclosan target gene due to the acquisition of an additional fabI allele derived from Staphylococcus haemolyticus (sh-fabI). In the current work, the genomic sequencing of 10 of these strains allowed the characterization of two novel composite transposons TnSha1 and TnSha2 involved in the spread of sh-fabI. TnSha1 harbors one copy of IS1272, whereas TnSha2 is a 11.7 kb plasmid carrying TnSha1 present either as plasmid or in an integrated form generally flanked by two IS1272 elements. The target and mechanism of integration for IS1272 and TnSha1 are novel and include targeting of DNA secondary structures, generation of blunt-end deletions of the stem-loop and absence of target duplication. Database analyses showed widespread occurrence of these two elements in chromosomes and plasmids, with TnSha1 mainly in S. aureus and with TnSha2 mainly in S. haemolyticus and S. epidermidis. The acquisition of resistance by means of an insertion sequence-based mobilization and consequent duplication of drug-target metabolic genes, as observed here for sh-fabI, is highly reminiscent of the situation with the ileS2 gene conferring mupirocin resistance, and the dfrA and dfrG genes conferring trimethoprim resistance both of which are mobilized by IS257. These three examples, which show similar mechanisms and levels of spread of metabolic genes linked to IS elements, highlight the importance of this genetic strategy for recruitment and rapid distribution of novel resistance mechanisms in staphylococci

Statistical analysis: number of blood cultures with one, two or three pneumococcal variants observed at 24 h in the experimental infection compared to those predicted from the statistical analysis.

<p>Statistical analysis: number of blood cultures with one, two or three pneumococcal variants observed at 24 h in the experimental infection compared to those predicted from the statistical analysis.</p