Simone Cagnacci

European Emergence of Ciprofloxacin-Resistant Escherichia coli Clonal Groups O25:H4-ST 131 and O15:K52:H1 Causing Community-Acquired Uncomplicated Cystitis

ABSTRACT A total of 148 E. coli strains displaying reduced susceptibility to ciprofloxacin (MIC ≥ 2 μg/ml) and causing uncomplicated urinary tract infections in eight European countries during 2003 to 2006 were studied. Their phylogenetic groups, biochemical profiles, and antibiotic susceptibilities were determined. Determination of the O:H serotype, pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD) PCR, and multilocus sequence typing provided additional discrimination. The majority (82.4%) of the microorganisms (122/148) carried resistance to two or more additional drugs, with the pattern ciprofloxacin-trimethoprim-sufamethoxazole-tetracycline-ampicillin being the most represented (73 strains out of 148; 49.3%). Extended-spectrum beta-lactamase production was detected in 12/148 strains (8.1%), with CTX-M-15 being the most-common enzyme. Six strains out of the whole collection studied (4.0%) contained a qnrB-like gene. Overall, 55 different PFGE or RAPD PCR profiles could be distinguished, indicating a substantial heterogeneity. However, about one-third (51/148) of the strains belonged to two clonal groups: O15:K52:H1 (phylogenetic group B2, lactose-nonfermenting variant, ciprofloxacin MIC of 16 μg/ml) and O25:H4 sequence type 131 (ST-131) (phylogenetic group D, ciprofloxacin MIC of ≥32 μg/ml). With the exception of Poland, strains of these two groups were isolated in samples from all participating countries but more frequently in samples from Spain and Italy. In some representative strains of the two main clonal groups, alterations in GyrA and ParC were the basic mechanism of fluoroquinolone resistance. In some members of the O25:H4 ST-131 group, displaying a ciprofloxacin MIC of >32 μg/ml, additional OmpF loss or pump efflux overexpression was found. In the Mediterranean area, strains belonging to these two clonal groups played a major role in determining the high rate of fluoroquinolone-resistant E. coli strains observed in the community.

Alteration of Membrane Permeability of Bacteria and Yeast by High Frequency Alternating Current (HFAC)

Aims: Endox® Endodontic System (Endox) is used for endodontic treatment by a high frequency alternating current (HFAC). This device damaged the envelopes of spores and vegetative organisms. If the integrity of the envelope is compromised, the transit of compounds in the two directions is possible. This latter aspect was investigated here. Methods: The instrument delivered a 60ms pulse at a frequency 300 kHz, and power 800 KV/m. DNA transfer was verified using Escherichia coli K-12 strain carrying a non conjugative plasmid pBP517 (gyrA+) as donor and a rifampicin and nalidixic acid resistant recipient. 0.2 ml of mixture of donor and recipient strains in saline was exposed to HFAC and plated on selective media. Uptake of antimicrobials and a delay in re-growth was assessed exposing the strains to HFAC. Results: Plasmid transfer was detected under different experimental conditions. From 9 to 27 recombinants were found. Representative recombinants cured from plasmid showed the original phenotype. HFAC promoted the uptake of ineffective antibiotics, and induces a 1 h of delay in re-growth on the strains. Conclusions: Endox exhibited an effect on microrganisms which is reminiscent with that occuring in electroporation, but with a mode of action that saved materials and time.

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.

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.

In vitro interaction between ceftazidime and vancomycin/teicoplanin in the presence of azithromycin againstPseudomonas 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.