Simona Roveta

Effects of rifaximin on bacterial virulence mechanisms at supra- and sub-inhibitory concentrations.

Abstract Rifaximin, a poorly absorbed rifamycin derivative, exhibited time-dependent bactericidal activity and at concentrations as low as 1/32 of the minimum inhibitory concentration (MIC) caused morphological alterations in both susceptible and resistant bacterial strains. Spontaneous rifaximin-resistant clones appeared with an incidence of 2.6×10−7. The percentage of Escherichia coli cells cured of various plasmids ranged from: 4.5-70% (Flac), 0-18% (pBP507), 7.7-43.8% (plasmid carrying ESBL genes) and 22.4-41.6% (plasmid encoding toxin from ETEC mex264). 8.4-18.2 and <0.1- 18% of Staphylococcus aureus cells were cured (plasmid-mediated penicillinase), 9.5-58.6% of Morganella morganii (ESBL), 10.6-47.1% Citrobacter freundii(ESBL), 2.3-38.7% of Proteus mirabilis (ESBL) and 14.3-66.6% of Klebsiella pneumoniae (ESBL). Rifaximin reduced plasmid transfer from donor to recipient strains by >99%. The MIC of ceftazidime was reduced (2-4 dilutions) in the presence of rifaximin (0.5xMIC) in ESBL producing strains. Rifaximin lowered the viability and virulence of the bacteria even though they developed resistance to the compound. In conclusion, the present findings add new features to the microbiological characteristics of rifaximin and suggest that if in vivo pathogens are exposed to sub-MICs of the drug, not only are their physiological functions compromised, but gene virulence and antibiotic resistance are not fully expressed.

Activity of daptomycin on biofilms produced on a plastic support by Staphylococcus spp.

The aim of this study was to assess whether the novel lipopeptide daptomycin might be capable of disrupting or inhibiting the synthesis of biofilms produced by staphylococci. Fourteen recently isolated slime-producing methicillin-susceptible (MET-S) and methicillin-resistant (MET-R) strains (three MET-S Staphylococcus aureus, three MET-R S. aureus, three MET-S Staphylococcus epidermidis, three MET-R S. epidermidis and two vancomycin-intermediate S. aureus (VISA)) were tested. Slime formation on polystyrene plates was quantified spectrophotometrically. Daptomycin (2-64 mg/L) inhibited slime synthesis by > or =80% in MET-S strains, by 60-80% in MET-R S. aureus and by 70-95% in MET-R S. epidermidis. At 64 mg/L, biofilm synthesis decreased by 80% in the VISA isolates. Daptomycin also disrupted pre-formed biofilm: >50% breakdown of initial biofilm (5h) was observed in all strains. Disruption of mature biofilms (48 h), in terms of percentage, was more variable depending on the strain, ranging from ca. 20% in a MET-R S. epidermidis strain to almost 70% in two MET-S strains (one S. aureus and one S. epidermidis). Daptomycin at concentrations achievable during therapy promoted a statistically significant inhibition of slime synthesis (preventing biofilm building) and induced slime disruption (disaggregating its structure) both in initial and mature biofilms on a plastic support in all staphylococcal strains studied.

Evaluation of the Uro-Quick, a new rapid automated system, for the detection of well-characterized antibiotic-resistant bacteria.

Abstract The Uro-Quick system has been employed to detect antibiotic resistance ingenotypically and/or phenotypically well-characterized bacterial species includingthose that might not be easily identified by routine procedure. In order to achievefull agreement between the antibiotic susceptibility results obtained by the reference method (NCCLS) and the Uro-Quick system, the optimal experimental conditions (inoculum size, time of incubation and antibiotic concentration) for each strain to be used by the automatic system were determined. The shorter time periods for generation of correct susceptibility results were 180 min for ampicillin- and ciprofloxacin-resistant Escherichia coli and for ESBL- and Inhibitor-resistant TEM (IRT)-producing E. coli; 360 min for penicillin-susceptible Streptococcus pneumoniae, as well as for strains with reduced susceptibility to this antibiotic (both intermediate, and resistant isolates). The same time was required to detect erythromycin-resistant pneumococci irrespective of their mechanism of resistance (ribosomal methylation and efflux-mediated), Streptococcus pyogenes exhibiting the three erythromycin-resistance phenotypes (constitutive, inducible and M-type) and Klebsiella pneumoniae, Enterobacter aerogenes, Enterobacter cloacae, Serratia marcescens, Proteus mirabilis and Moraxella morganii refractory to third-generation cephalosporins, aminoglycosides, ciprofloxacin and other classes of antimicrobial agents; 480 min for penicillin-resistant, constitutive and inducible oxacillinresistant (OXA-R) Staphylococcus aureus and OXA-R Staphylococcus epidermidis. The same period of time was also necessary to find the great majority of drug-resistance exhibited by Pseudomonas aeruginosa. Teicoplanin-resistant Staphylococcus haemolyticus, vancomycin-resistant (VanA, VanB, VanC) highlevel aminoglycoside-resistant (HLAR) Enterococcus spp, and imipenem-resistant P. aeruginosa required longer incubation (24 h) to be detected. The results obtained indicate that Uro-Quick might be a reliable and promising instrument for the correct detection of the above antibiotic resistance markers.

Tigecycline in vitro activity against gram-negative and gram-positive pathogens collected in Italy.

Background: In this study (part of the global TEST program), the in vitro activity of tigecycline, a member of a new class of antimicrobial agents, the glycylcyclines, against clinical isolates collected in Italy was evaluated. Methods: A total of 200 clinical isolates were collected and identified in our institution during 2005. Minimum inhibitory concentrations (MICs) of the antimicrobial agents were determined by the broth microdilution method recommended by the CLSI in 2005. Results: Globally, 135 Gram-negative and 65 Gram-positive pathogens were evaluated. Tigecycline demonstrated excellent inhibitory activity against Acinetobacter spp., Haemophilus influenzae, Escherichia coli, Enterococcus spp., Staphylococcus aureus, Streptococcus agalactiae and Streptococcus pneumoniae with MIC90 ≤1 mg/l. Conclusion: Tigecycline exhibited potent in vitro antibacterial activity (comparable to or greater than most commonly employed antimicrobials) against both Gram-positive and Gram-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.

Antibiotic susceptibility tests directly on urine samples using Uro-Quick, a rapid automated system.

Abstract During the period June 2003-March 2004, 12,579 urine samples were examined employing the Uro-Quick system. Positive samples (1,948) were subsequently Gram-stained and processed by standard procedures for microorganism identification and antibiotic susceptibility determination by the disk diffusion method. Results of this latter test were compared with those obtained employing the new rapid Uro-Quick method. Antibiotics were introduced in vials containing 2 ml of Mueller-Hinton broth, then 0.5 ml of urine or a bacterial suspension in broth were added; a vial without drug was used as control. After 3 and 5 hours of incubation (for Gram-negative and Gram-positive strains respectively) the instrument showed the results. No growth and a growth curve like the control indicated susceptible and resistant strains respectively. Overall 1,590 Gram-negative strains were tested against ciprofloxacin, nitrofurantoin, amoxicillin-clavulanate, ceftazidime, fosfomycin, imipenem, amikacin, trimethoprim-sulfamethoxazole, and piperacillin-tazobactam, while 358 Gram-positive bacteria were assessed against ciprofloxacin, nitrofurantoin, amoxicillin-clavulanate, ampicillin, fosfomycin, gentamicin, oxacillin and trimethoprim-sulfamethoxazole. Against the major urinary tract pathogens (Escherichia. coli, enterococci, Klebsiella spp. and Proteus spp.) agreement between the Uro-Quick system and the disk diffusion test generally was >90% for all antibiotics tested. On the basis of these results the system appears useful not only for bacteriuria screening, but also to rapidly test the antibiotic susceptibility of common uropathogens.

In vitro activity of ceftibuten at sub-inhibitory concentrations in comparison with other antibiotics against respiratory and urinary tract pathogens.

Abstract Some new features of the In Vitro activity of ceftibuten, an oral third generation cephalosporin, have been studied in reference to respiratory and urinary tract pathogens included in its antibacterial spectrum. At 0.25XMIC (minimum inhibitory concentration) and 0.5XMIC levels, ceftibuten was able to affect the biofilm production in 2/3 of both Escherichia coli and Proteus mirabilis strains, and reduced the number of strains capable of adhering to epithelial cells by about 35% in comparison to the control. Surface hydrophobicity was also influenced by ceftibuten and the other drugs at 0.25-0.5XMIC. In general, no marked variation in the virulence traits of the pathogens studied were found by exposing bacteria to sub-MICs of ceftibuten. Plasmid loss (from 1.8 to 37.2%), and Flac transfer inhibition (about 30-50% reduction in the number of recombinants) were detected under the experimental conditions used. This study confirms the excellent antibacterial properties of ceftibuten by adding new information about the effects of this antibiotic against pathogens often involved in respiratory and urinary tract infections that may be treated with this compound, supporting the appropriate use of this cephalosporin.

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.