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

Simple Records Matching Method for diagnostic and clinical datasets of patient’s records

Several statistical packages, either commercials or open-source, provide many methods for multi-factorial and discriminant analysis; such a software is poorly used by physicians. Appropriate models and tests have to be used pending on the kind of experiment scheme, adequate distribution assumption are needed for variables and parameters and proper data validation have to be verified for historical records. These are but a few of many critical aspects for a robust and trustable data interpretation needed in the Evidence Based Medicine era. Clinicians always wish to be able to quickly interpreter diagnostic records to discriminate, or alternatively correlate, coherent groups of patient’s records according to either descriptive characters or variable units. Practically, patient’s records are stored in spread-sheet or database which change pending on the clinical trial scope; moreover, data entry and its validation is usually poor, hence physician are used to send raw-data to the statistician without contributing, for instance, with parametric and non-parametric indication on usable distribution. We address this problem by introducing a simple “weighted” model approached with the Unique Factorisation Domain theory: records can be compare by matching each other through a score overlap and clinician can modulate tolerance of closeness stringency criteria. An intuitive paradigm of records matching method (RMM) is presented and discussed with example, computational design and programming prototyping model; freely available material concerning real-world application, are also provided by the authors

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

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

Impiego del sistema URO-QUICK per l'esecuzione rapida di antibiogrammi direttamente su campioni di urine

n/

In vitro antimicrobial activity of tigecycline against Gram negative and Gram positive pathogens collected in Northen Italy (T.E.S.T. program 2010)

Background. In this study (part of the global T.E.S.T. program) was evaluated the in vitro activity of tigecycline, member of a new class of antimicrobial agents, the glycylcyclines, against clinical isolates collected in Italy. Methods. A total of 194 clinical isolates were collected and identified in our Institution during 2010. Minimum inhibitory concentrations (MICs) of the antimicrobial agents were determined by the CLSI (2010) recommended broth microdilution method. Results. Globally 129 Gram negative and 65 Gram positive pathogens were evaluated.Tigecycline demonstrated excellent inhibitory activity against Escherichia coli, Haemophylus influenzae, Enterococcus spp., Staphylococcus aureus MetS, Streptococcus pneumoniae and Streptococcus agalactiae with MIC90 1mg/l. Conclusion. Tigecycline exhibited potent in vitro antibacterial activity (comparable to or greater than most commonly employed antimicrobials) against both Gram positive and negative clinical pathogens. These data suggest that tigecycline, with an expanded broad-spectrum antimicrobial activity, may be an effective empiric therapeutic option for the treatment of serious infections caused by clinically relevant pathogens

Characterization of colistin-resistant A. baumannii isolated in Intensive Care Unit of an Italian Hospital

We report the characterization of an Acinetobacter baumannii resistant to colistin isolated from a patient treated with colistin for 22 days. The identification and initial susceptibility testing of the strain was performed at ASL3 Imperiese with the Vitek-2 automated system and than the strain was re-identified at the Sezione di Microbiologia with APINE. In vitro activity of antimicrobial agents was determined by the microdiluition methods. The detection of the beta-lactamase gene was performed by PCR and for the analysis of LPS were sequenced the genes of the PmrABC system. We compared the PFGE profile of the colistin-resistant A. baumannii with 5 A. baumannii colistin-susceptible strains isolated in the same Hospital during 2010. The strain was resistant to all antimicrobial agents tested, except to tygecyclin. The PFGE shown that the A. baumannii colistin-resistant was closery related to the colistin-susceptible strains. The sequencing of the PmrABC system showed that the strain had a single mutation in the PmrB component. Colistin-resistant strain have recently been found in several Gram-negative bacteria such A. baumannii, K. pneumoniae and P. aeruginosa. This clinical case confirms that resistance to colistin in A. baumannii can be selected in vivo following the use of colistin in therapy

Evaluation of the Uro-Quick system for antibiotic susceptibility tests of strains collected from intensive care units

During the period January–June 2004, 525 pathogens isolated from intensive care units were examined with the new rapid Uro-Quick method for antibiotic susceptibility tests. The results were compared with those obtained by the reference NCCLS methods (disk diffusion or dilution). Antibiotic (in appropriate concentration) was introduced in a vial containing 2 ml of Mueller-Hin ton broth, then 0.5 ml of 5×10 or 106 cells/ml of the strain culture were added. After 3–6 h of incubation, depending on the microorganism studied, the instrument printed the results: no growth and a growth curve similar to that of the untreated control are representative of a susceptible and resistant strain respectively. The following drugs were tested: ciprofloxacin, ampicillin, aztreonam, co-clavulanate, piperacillin/tazobactam, ceftazidime, cefotaxime, cefuroxime, ceftriaxone, imipenem, amikacin, gentamicin, trimethoprim-sulfamethoxazole, clindamycin, erythromycin, linezolid, penicillin, tetracycline, vancomycin, oxacillin. Gram-negative strains tested were 252 and Gram-positive 273: agreement between the two methods ranged from 85.6% (piperacillin/tazobactam) to 98.5% (ciprofloxact) in Gram-negative pathogens, from 90 to 100% in Gram-positive, with the exception of erythromycin (84.2%) against enterococci. On the basis of the present findings the Uro-Quick system appears to be very useful for the rapid detection of antibiotic susceptibility in pathogens collected from intensive care units