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

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 x 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 (0.5 x MIC) 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.

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 MIC(90)

Activity of moxifloxacin on biofilms produced in vitro by bacterial pathogens involved in acute exacerbations of chronic bronchitis.

The aim of this study was to assess whether moxifloxacin is able to inhibit the synthesis of and to disrupt biofilms produced in vitro by bacterial pathogens involved in acute bacterial exacerbations of chronic bronchitis. Three strains each of Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis, Staphylococcus aureus and Escherichia coli recently isolated from clinical respiratory specimens and capable of slime production were used. Biofilm formation on polystyrene plates was quantified spectrophotometrically by established methodologies. Moxifloxacin (0.5 mg/L) inhibited slime synthesis by >70% in S. aureus, H. influenzae and S. pneumoniae, 45-70% in E. coli and 35-70% in M. catarrhalis. Disruption of pre-formed structures was also promoted by moxifloxacin both for initial (5h) and mature (48 h) biofilms. Drug concentrations reached during therapy (0.5-4 mg/L) resulted in a breakdown of initial biofilm of 60-80% in H. influenzae and S. pneumoniae, 48-86% in S. aureus, 37-69% in M. catarrhalis and 51-71% in E. coli. Mature biofilms were less susceptible to degradation. Moxifloxacin at concentrations that can be achieved in the bronchial mucosa during therapy therefore promotes a significant inhibition of biofilm synthesis and induces slime disruption, a feature that may be instrumental in reducing the exacerbations so frequently observed in this condition.

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

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.

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

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.

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

The Uro-Quick system has been employed to detect antibiotic resistance in genotypically and/or phenotypically well-characterized bacterial species including those that might not be easily identified by routine procedure. In order to achieve full 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 oxacillin-resistant (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) high-level 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.

Antibiotic persistence: the role of spontaneous DNA repair response.

Persisters are a small proportion of a bacterial population that exists in a physiological state permitting survival despite the lethal activity of antibiotics. To explain this phenomenon, it has been suggested that persisters are bacteria repairing spontaneous errors of DNA synthesis. To verify this assumption, Escherichia coli AB1157 and its lexA3 derivative were exposed to a dose 6x MIC of various antibiotics representative of different molecular mechanisms of action (ampicillin, ceftriaxone, meropenem, amikacin, ciprofloxacin). Bacterial cell counts, after 24 hr of exposure to the antimicrobials, revealed a reduction of about 90% of viable organisms in the lexA3 strains in comparison to the lexA+. In several cases, the number of colony-forming units decreased below the limit of assay. This behavior was noted with all antibiotics used, alone or in combination (amikacin plus ceftriaxone and amikacin plus ciprofloxacin). The same experiments were repeated using E. coli AB1157 cultured in the presence of mitomycin C (0.25x MIC), and the number of survivors exceeded by about 90% the values found in the nonexposed control. In contrast, in the sulA background, mitomycin C reacted synergically with all the antibiotics tested causing a strong reduction of the survivors in comparison with the control. The addition of chloramphenicol (0.125x MIC), on the contrary, caused a reduction of the number of survivors of about 90%. These findings indicate that, when DNA repair is active (a mechanism known to block cell division), the number of survivors is greater than that observed with lexA3. Thus, in addition to other possible explanations, persisters might be a fraction of bacteria that during antibiotic treatment are not growing because they are repairing spontaneous errors of DNA synthesis.

[Epidemiology of methicillin-resistance among Staphylococci strains isolated in risk units and effects of the vancomycin on the expression of methicillin-resistance]. / Epidemiologia della meticillino-resistenza negli Stafilococchi isolati in reparti a rischio ed effetti della vanomicina sulla espressione della meticillino-resistenza.

Four hundred Staphylococci strains, isolated from different in intensive care unit hospitalized patients, were analyzed. 53% of all strains were resistant to methicillin. Against methicillin-resistant S. aureus (MRSA), teicoplanin and vancomycin (100% of susceptibility), rifampin (76.3%) and co-trimoxazole (73%) emerged as the most potent drugs tested; the 15% of the strains were susceptible to ciprofloxacin, erythromycin, clindamycin and gentamicin. Only one MRSA strain (0.8%) resulted hetero-resistant to vancomycin. Among 100 strains exposed to serial concentration of vancomycin (0.25-32 mg/L for 30 days), 57 were selected with intermediate-level of resistance to the glycopeptides; the MRSA strains have shown to acquire resistance in vitro more easily than methicillin-susceptible. These results indicate that in the clones of Staphylococci circulating in our region, the evolution of glycopeptides-resistance is not a rapid process and the loss of effectiveness of these antibiotics cannot be predicted to short term. In particular, the restriction profile analysis of chromosomal DNA from MRSA strains, selected in vitro with intermediate -level of vancomycin resistance, demonstrated that at the moment in the hospital departments studied, the diffusion of a clone able to acquire resistance more easily than others is not present.