The role of gyrA , gyrB , and dnaA functions in bacterial conjugation

The role of DNA gyrase in F'lac plasmid conjugation was studied using Escherichia coli gyrA43 (Ts), gyrB41(Ts), and dnaA46(Ts) thermosensitive mutants as donor or recipient organisms, and a rifampicin or nalidixic acid-resistant J-53 strain in the presence or absence of nalidixic acid. Mating experiments were also performed employing Hfr derivatives of the thermosensitive strains. Conjugation was carried out in broth for 60 min using a standard method at permissive and non-permissive (32 and 43 °C) temperatures, with or without drugs. At 32 °C, nalidixic acid reduced the number of transconjugants by about 97 % in comparison to the control, while at 43 °C, the drug inhibited F'lac transfer by about 98 % from dnaA46(Ts) mutant and by about 6.5 % from gyrA43(Ts) and 15 % from gyrB41(Ts) hosts. Using the temperature-sensitive mutants as recipient strains, the transconjugants found were approximately the same under all conditions. The number of transconjugants did not change significantly when nalidixic acid-resistant strains were used as donor or recipient strains. Lastly, nalidixic acid reduced the number of transconjugants from Hfr selected in the above mutants under all experimental conditions. These findings suggest that F'lac transfer does not involve DNA gyrase activity

Does the adoption of EUCAST susceptibility breakpoints affect the selection of antimicrobials to treat acute community-acquired respiratory tract infections?

Background: In several European Countries, by the end of 2012, CLSI guidelines will be replaced by EUCAST. We compared antimicrobial susceptibility results of a large number of respiratory pathogens using both EUCAST and previously adopted CLSI criteria to evaluate the impact on susceptibility patterns and the possible consequences that could occur in clinical practice due to this replacement.For S. pyogenes and S. aureus, the interpretation of susceptibility data using the EUCAST criteria did not produce relevant changes in comparison to CLSI.Against S. pneumoniae, more restrictive EUCAST breakpoints could lead to increased benzylpenicillin and/or amoxicillin-clavulanate resistance rates, which in turn could translate in increased dosages of these antibiotics or usage of alternative agents for respiratory tract infections.Against S. pneumoniae, M. catarrhalis and H. influenzae, cefuroxime-axetil and cefaclor produced the most divergent results depending on the breakpoints adopted and these striking differences could lead to the revision of those guidelines suggesting these two cephalosporins as alternatives in the management of upper respiratory tract infections.Discussion: Many differences exist between CLSI and EUCAST breakpoints. However, only in a few cases do these differences translate in major interpretive category discrepancies. In countries adopting more restrictive EUCAST breakpoints, clinicians should be aware of these discrepancies and that they could be faced with antibiotic-resistant respiratory pathogens more frequently than before.Summary: The interpretive discrepancies between EUCAST and CLSI suggest that the discussion on the management of community-acquired respiratory tract infections is still open and further studies are desirable to better define the role of some antibiotic

In vitro interaction between ceftazidime and vancomycin/teicoplanin in the presence of azithromycin against Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen, intrinsically resistant to many antibiotics and prone to acquire resistance against many drugs. It is assumed that agents that disorganise the structure of the outer membrane might allow the passage of other drugs into cell. To verify this hypothesis, ceftazidime (CAZ) has been tested in association with glycopeptides (GLYs) and azithromycin (AZI). Time-kill experiments were performed on a representative strain. CAZ in combination with GLYs showed 99, 90 and 10% of CFU/ml reduction in 33.9,52.5 and 13.6% of the cases, respectively; the addition of AZI increased the incidence of 99% CFU/ml reduction to 42% of the cases. Indifference was the most common finding, and additive/synergism in the other cases. Present findings demonstrated that CAZ favourably reacted with GLYs in the presence of AZI

Diversity Assessment and DNA-Based Fingerprinting of Sicilian Hazelnut (Corylus avellana L.) Germplasm

The characterization of plant genetic resources is a precondition for genetic improvement and germplasm management. The increasing use of molecular markers for DNA-based genotype signature is crucial for variety identification and traceability in the food supply chain. We collected 75 Sicilian hazelnut accessions from private and public field collections, including widely grown varieties from the Nebrodi Mountains in north east Sicily (Italy). The germplasm was fingerprinted through nine standardized microsatellites (SSR) for hazelnut identification to evaluate the genetic diversity of the collected accessions, validating SSR discrimination power. We identified cases of homonymy and synonymy among acquisitions and the unique profiles. The genetic relationships illustrated by hierarchical clustering, structure, and discriminant analyses revealed a clear distinction between local and commercial varieties. The comparative genetic analysis also showed that the Nebrodi genotypes are significantly different from the Northern Italian, Iberian, and Turkish genotypes. These results highlight the need and urgency to preserve Nebrodi germplasm as a useful and valuable source for traits of interest employable for breeding. Our study demonstrates the usefulness of molecular marker analysis to select a reference germplasm collection of Sicilian hazelnut varieties and to implement certified plants’ production in the supply chain

Characterization of high frequency of recombination strains selected by integrative suppression of F'lac in dnaA, gyrA and gyrB temperature sensitive mutants

Integration of F'lac plasmid into chromosome of both gyrA(Ts) and gyrB(Ts) cells phenotypically suppress the thermosensitive mutations of the DNA gyrase enzymes. As the comparative strains isolated from dnaA(Ts), these high frequency of recombination (Hfr) derivatives were able to transfer chromosomal markers to recipient strains, showed a growth rate of about 60 min, and developed filamentous forms when incubated at the temperature of 43°C. Conversely to dnaA(Ts) Hfr selected isolates, the great majority of Hfr derivative of gyrase mutants resulted resistant to acridine orange and rifampin. Time-kill experiments carried out at the non-permissive temperature also revealed that nalidixic acid has no antibacterial activity on these Hfr strains while derivatives of dnaA(Ts) mutant, as well as the control strain HfrH, were strongly inhibited by this drug. Therefore F plasmid induced duplication of chromosome in the mutants even if the DNA gyrase enzymes are not working. Of a certain interest is that these bacteria exhibit physiological perturbations that affect the main cellular functions, however, they do not appear essential for the survival of the strains

direct transfer of genetic material between two escherichia coli k12 strains by electroporation

A direct transfer of plasmid pBP517 from C600 to J53 Escherichia coli K12 strains by electroporation, either by the standard method, or in the presence of 20 μg/mL of DNAse was carried out. In a standard experiment, donor and recipient bacteria were mixed and subjected to the electroporation procedure, about 2700 (range 1600-3700) recombinants were found, while no colonies were detected from non treated bacteria. When the same tests were performed at the presence of DNAse the number of recombinants fell to about 200 (range 183-218). This difference between the number of colonies found in the presence or in absence of DNAse was observed every time the tests were repeated. According to these observations, plasmid DNA has been transferred from donor to recipient cells via electroporation also when DNAse was added. Since the free genetic material is destroyed by the enzyme and recombination takes place it has been hypothesized that there must be a direct contact between the partner cells

isolation of an escherichia coli mutant susceptible to a quinolone in an anaerobic environment

Quinolones are bactericidal agents that interfere with the essential prokaryotic enzyme DNA gyrase. While their mechanism of killing appears to be elucidated, one interesting feature is represented by the fact that, under anaerobic conditions, the growth of bacteria is inhibited but their viability is not affected by the first generation of quinolones such as nalidixic acid. More information about the mode of action of these drugs in anaerobiosis might be gained through the availability of strains subjected to enhanced killing in oxygen-deprived media. It has been assumed that when a population of a AB1157(F'lac) strain is exposed to nalidixic acid, plasmid-free cells could be recovered from culture treated with sub-inhibitory concentrations of the drug (2 mg/L) in aerobiosis, and, at the same drug level, only from the rare spontaneous susceptible mutant(s) in anaerobiosis. Among plasmid free bacteria found, 1 isolate demonstrated the same MIC value to nalidixic acid in both aerobic and anaerobic conditions. The mutation was co-transferred with Tn10 inserted at 28.5 min of the Escherichia coli genetic map into a wildtype strain. These transductants revealed the same phenotypes of the original mutant: susceptibility to nalidixic acid under anaerobic conditions (assessed by time-kill tests) and elongated cells during the aerobic growth, generation time about 65 min in comparison to 25 min of the control. Time kill experiment under aerobic environment revealed that the transductant was also susceptible to ciprofloxacin but not nalidixic acid in the presence of chloramphenicol (50 mg/L). These results suggest a possible role of bacterial topoisomerase in the anaerobic susceptibility to nalidixic acid of the mutant

DNMT3B in vitro knocking-down is able to reverse embryonal rhabdomyosarcoma cell phenotype through inhibition of proliferation and induction of myogenic differentiation

Aberrant DNA methylation has been frequently observed in many human cancers, including rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children. To date, the expression and function of the de novo DNA methyltransferase (DNMT) 3B in RMS have not yet been investigated. Our study show for the first time a significant up-regulation of DNMT3B levels in 14 RMS tumour samples and 4 RMS cell lines in comparison to normal skeletal muscle. Transfection of RD and TE671 cells, two in vitro models of embryonal RMS (ERMS), with a synthetic DNMT3B siRNA decreased cell proliferation by arresting cell cycle at G1 phase, as demonstrated by the reduced expression of Cyclin B1, Cyclin D1 and Cyclin E2, and by the concomitant up-regulation of the checkpoint regulators p21 and p27. DNMT3B depletion also impaired RB phosphorylation status and decreased migratory capacity and clonogenic potential. Interestingly, DNMT3B knock-down was able to commit ERMS cells towards myogenic terminal differentiation, as confirmed by the acquisition of a myogenic-like phenotype and by the increased expression of the myogenic markers MYOD1, Myogenin and MyHC. Finally, inhibition of MEK/ERK signalling by U0126 resulted in a reduction of DNMT3B protein, giving evidence that DNMT3B is a down-stream molecule of this oncogenic pathway.Taken together, our data indicate that altered expression of DNMT3B plays a key role in ERMS development since its silencing is able to reverse cell cancer phenotype by rescuing myogenic program. Epigenetic therapy, by targeting the DNA methylation machinery, may represent a novel therapeutic strategy against RMS

The Susceptibility of Candida albicans to Gamma-Radiations and Ketoco-nazole Depends on Transitional Filamentation

The virulence of C. albicans is associated with the transitional evolution from yeast to filamentous forms. We were interested in the effects amphotericin B (AMB), ketoconazole (KTC) and γ-radiations might have on these broadly defined phenotypes as determined by the CFU procedure. By using collagen gel as the 3-dimensional support of cell culture, diverse experimental conditions were contemplated in order to modulate the differentiation of Candida during sessile and planktonic growth. These conditions included the co-culture with human epithelial and endothelial cells and treatment with farnesol, tyrosol and conditioned medium from P. aeruginosa. The overall results were as follows: 1) The survival of Candida was inhibited by the exposure to γ-radiations, but only after the organism was induced to progress into excess filamentation, while in normal growth conditions it proved to be radioresistant; 2) AMB inhibited the growth of yeast forms, while KTC was specifically toxic to filamentous forms and 3) the combined treatment of filamentous Candida with KTC and γ-radiations resulted in the synergistic inhibition of the organism. These findings indicate that both the radiosensitivity of C. albicans and its response to the synergistic effects of γ-radiations and KTC are filamentation-dependent pharmacological processes