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Spreading of resistance to antibiotics is of great concern due to the increasing rate of isolation of multiresistant pathogens. Since commensal bacteria may transfer determinants of resistance to pathogens, studies on development of resistance should include also lactobacilli. Resistance to macrolides, penicillins and tetracycline was determined in 40 isolates of Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus crispatus, and Lactobacillus casei isolated from faeces of apparently healthy volunteers. Frequency of mutation and changes in susceptibility after serial exposure to these antibiotics at concentrations of 4× and 8× MIC were evaluated in susceptible isolates. Acquired resistance was defined as an increment in MIC values of at least four times in respect to the pre-selection values. Resistance to macrolides and/or tetracycline was identified in 14 and 4 isolates, respectively. ermB gene and A2058G mutation in 23S rRNA were detected in macrolide resistant isolates. Frequencies of mutation of susceptible isolates (n=26) were lower for ampicillin and erythromycin than for tetracycline. Serial exposure to antibiotics led to selection of resistant mutants. However, acquired resistance was rather unstable and was lost after subcultures in antibiotic-free medium in most mutants. Resistance to erythromycin was associated to a A2058G mutation in 23S rRNA. In conclusion, results indicate that resistance to macrolides and tetracycline is present among intestinal lactobacilli. Decrease in susceptibility following serial exposure to antibiotics might occur in lactobacilli, in a strain- and antibiotic-dependent way. Since lactobacilli are often used as probiotics, their ability to acquire resistance should be evaluated for isolates candidate to be included in probiotics based products.

Background: Fluoroquinolones are potent antimicrobial agents used for the treatment of a wide variety of community- and nosocomial- infections. However, resistance to fluoroquinolones in Enterobacteriaceae is increasingly reported. Studies assessing the ability of fluoroquinolones to select for resistance have often used antimicrobial concentrations quite different from those actually acquired at the site of infection. The present study compared the ability to select for resistance of levofloxacin, ciprofloxacin and prulifloxacin at concentrations observed in vivo in twenty strains of Escherichia coli and Klebsiella spp. isolated from patients with respiratory and urinary infections. The frequencies of spontaneous single-step mutations at plasma peak and trough antibiotic concentrations were calculated. Multi-step selection of resistance was evaluated by performing 10 serial cultures on agar plates containing a linear gradient from trough to peak antimicrobial concentrations, followed by 10 subcultures on antibiotic-free agar. E. coli resistant strains selected after multi-step selection were characterized for DNA mutations by sequencing gyrA, gyrB, parC and parE genes., Results: Frequencies of mutations for levofloxacin and ciprofloxacin were less than 10-11 at peak concentration, while for prulifloxacin they ranged from <10-11 to 10-5. The lowest number of resistant mutants after multistep selection was selected by levofloxacin followed by ciprofloxacin and prulifloxacin. Both ciprofloxacin- and prulifloxacin-resistant mutants presented mutations in gyrA and parC, while levofloxacin resistance was found associated only to mutations in gyrA., Conclusions: Among the tested fluoroquinolones, levofloxacin was the most capable of limiting the occurrence of resistance.

Background: Haemophilus influenzae is one of the main aetiological agents of community-acquired respiratory tract infections. The primary aim of this study was to evaluate the antibacterial activity of telithromycin against H. influenzae clinical isolates showing different pattern of resistance in comparison with azithromycin and clarithromycin at 1/4 x, 1/2 x, 1 x, 2 x, 4 x minimum inhibitory concentration (MIC) and to peak concentrations in epithelial lining fluid (ELF). The secondary aim was to determine the influence of CO2 enriched atmosphere on bacterial susceptibility., Results: Telithromycin showed high activity against H. influenzae, including strains susceptible to beta-lactams (n = 200), beta-lactamase producer (n = 50) and beta-lactamase negative ampicillin resistant (BLNAR) (n = 10), with MIC from < or =0.03 to 4 mg/L, and MIC50/MIC90 of 1/2 mg/L with susceptibility rate of 100%, and minimum bactericidal concentrations (MBC) from 2 to 4-fold higher than the MIC. Azithromycin was the most active tested macrolide (range: 0.25 - 4 mg/L; MIC50/MIC90: 1/2 mg/L), comparable to telithromycin, while clarithromycin showed the highest MICs and MBCs (range: 0.25 - 8 mg/L; MIC50/MIC90: 2/8 mg/L). In time-kill studies, telithromycin showed a bactericidal activity at the higher concentrations (4 - 2 x MIC and ELF) against all the strains, being complete after 12 - 24 hours from drug exposition. At MIC concentrations, at ambient air, bactericidal activity of telithromycin and azithromycin was quite similar at 12 hours, and better than that of clarithromycin. Besides, telithromycin and clarithromycin at ELF concentrations were bactericidal after 12 hours of incubation for most strains, while 24 hours were needed to azithromycin to be bactericidal. Incubation in CO2 significantly influenced the MICs and MBCs, and only slightly the in vitro killing curves., Conclusion: Telithromycin showed an in-vitro potency against H. influenzae comparable to azithromycin, with an in-vitro killing rate more rapid and superior to clarithromycin at 2X-MIC against beta-lactamase producers and BLNAR strains, and to azithromycin at ELF concentrations against beta-lactamase negative strains. Against all strains, MICs and MBCs were lower in the absence of CO2 for the tested antibiotics, showing an adverse effect of incubation in a CO2 environment. The in-vitro potency together with the tissue concentrations of the antimicrobial, should be considered in predicting efficacy.

Background: Methicillin resistant Staphylococcus aureus (MRSA) is an increasingly common cause of nosocomial infections, causing severe morbidity and mortality worldwide, and accounting in some hospitals for more than 50% of all S. aureus diseases. Treatment of infections caused by resistant bacterial pathogens mainly relies on two therapeutic modalities: development of new antimicrobials and use of combinations of available antibiotics. Combinations of antibiotics used in the empiric treatment of infections with suspected methicillin resistant Staphylococcus aureus etiology were investigated., Methods: Double (vancomycin or teicoplanin with either levofloxacin or cefotaxime) and triple (vancomycin or teicoplanin + levofloxacin + one among amikacin, ceftazidime, cefepime, imipenem, piperacillin/tazobactam) combinations were evaluated by means of checkerboard assay and time kill curves. Mutational rates of single and combined drugs at antimicrobial concentrations equal to the resistance breakpoints were also calculated., Results: Vancomycin or teicoplanin + levofloxacin showed synergy in 16/50 and in 9/50 strains respectively, while vancomycin or teicoplanin + cefotaxime resulted synergic for 43/50 and 23/50 strains, respectively. Triple combinations, involving teicoplanin, levofloxacin and ceftazidime or piperacillin/tazobactam gave synergy in 20/25 strains. Teicoplanin + levofloxacin gave synergy in triple combinations more frequently than vancomycin + levofloxacin. For single antibiotics, mutational frequencies ranged between 10(-5) and <10(-9) for levofloxacin, cefotaxime, amikacin and imipenem, and <10(-9) for vancomycin and teicoplanin. When tested in combinations, mutational frequencies fell below 10(-9) for all the combinations., Conclusion: In vitro evidence of synergy between glycopeptides, fluoroquinolones (levofloxacin) and beta-lactams and of reduction of mutational frequencies by combinations are suggestive for a potential role in empirical therapy of severe pneumonia with suspected MRSA etiology.

Objectives: The aim of this study was to evaluate the ability of levofloxacin and ciprofloxacin alone and in combination with either ceftazidime, cefepime, imipenem, piperacillin-tazobactam or amikacin to select for antibiotic-resistant mutants of Pseudomonas aeruginosa and Acinetobacter spp., Methods: Clinical strains of P. aeruginosa (n = 5) and Acinetobacter spp. (n = 5) susceptible to all the drugs used in the study were assayed. Development of resistance was determined by multi-step and single-step methodologies. For multi-step studies, MICs were determined after five serial passages on antibiotic-gradient plates containing each antibiotic alone or in combination with levofloxacin or ciprofloxacin. Acquisition of resistance was defined as an increase of >or=4-fold from the starting MIC. In single-step studies, the frequency of spontaneous mutations was calculated after a passage on plates containing antibiotics alone and in combinations at concentrations equal to the highest NCCLS breakpoints., Results: Serial passages on medium containing single antibiotics resulted in increased MICs for each antibiotic; MIC increases were limited by antibiotics in combination. A decrease in the number of strains with MICs above the NCCLS breakpoints occurred when fluoroquinolones were combined with a second antibiotic for both P. aeruginosa and Acinetobacter spp. isolates. Frequencies of mutation were higher for antibiotics alone than for combinations., Conclusions: Use of combinations of fluoroquinolones with beta-lactams and amikacin reduces the risk for in vitro selection of resistant P. aeruginosa and Acinetobacter spp.

Moxifloxacin (CAS 186286-86-8) is a recent fluoroquinolone addressed to the treatment of respiratory infections, although clinical efficacy has been shown also in the therapy of urinary tract diseases. The in vitro activity of moxifloxacin against one encapsulated strain of Klebsiella pneumoniae was determined by means of time-kill curves and by evaluation of effects of subinhibitory concentrations on bacterial morphology by means of scanning electron microscopy. Moreover, the efficacy and time requested to eradicate the same strain from lungs was determined after intratracheal infection of guinea pigs. K. pneumoniae was rapidly killed by moxifloxacin at concentrations greater than the minimum inhibitory concentration (MIC). Subinhibitory concentrations (1/4 and 1/8 of the MIC ) induced filamentation in a remarkable portion of bacteria. A progressive decrease in the bacterial content of lungs was observed during the 48-h period, with statistically significant differences compared with the control animals. In conclusion, this study provides in vivo evidence of the antimicrobial properties of moxifloxacin against K. pneumoniae.

Background: Combination therapy is used to widen the antimicrobial spectrum, minimize toxicity and prevent the emergence of resistant mutants., Methods: Synergy between levofloxacin or ciprofloxacin and ceftazidime, cefepime, imipenem, piperacillin-tazobactam and amikacin was evaluated by checkerboard assay with 55 strains and by time-kill curves with 8 strains of Pseudomonas aeruginosa and Acinetobacter spp., Results: In the checkerboard assay, synergy and additivity were the most frequent effects observed among all the combinations against P. aeruginosa and Acinetobacter spp., with no significant differences between the two fluoroquinolones. No antagonism was observed. In the time-kill curves, synergy was evidenced against all the tested strains, at least for one combination at one of the time points considered. Levofloxacin and ciprofloxacin combined with ceftazidime, as well as levofloxacin plus amikacin, were synergistic for all the strains tested., Conclusion: Combinations of fluoroquinolones with beta-lactams or amikacin show an enhanced activity against P. aeruginosa and Acinetobacter spp.

Objective: The in vitro abilities of telithromycin, azithromycin and clarithromycin to select for resistance were compared by testing isolates of Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae and beta-haemolytic streptococci., Methods: Five strains each of beta-lactamase-positive and beta-lactamase-negative H. influenzae, beta-lactamase-positive and beta-lactamase-negative M. catarrhalis, S. pneumoniae, beta-haemolytic group A, group C and group G streptococci and three strains of beta-lactamase-negative ampicillin-resistant H. influenzae were evaluated. Development of resistance was determined by multi-step and single-step methodologies. For multi-step studies, MIC values were determined after five serial passages on antibiotic-gradient plates and after 10 serial passages on antibiotic-free plates. Acquisition of resistance was defined as an increase of >/=4-fold from the starting MIC. In single-step studies, the rate of spontaneous mutations was calculated after a passage on plates containing antibiotics at concentrations equal to the highest NCCLS breakpoints., Results: Azithromycin, clarithromycin and telithromycin gave a >/=4-fold increase in 20, 20 and 10 streptococcus strains, in 4, 5 and 0 H. influenzae strains and in 2, 7 and 4 M. catarrhalis strains, respectively. After 10 passages on antibiotic-free plates, 21/26 strains for azithromycin, 22/32 for clarithromycin and 1/14 for telithromycin maintained high MIC values. In single-step studies, the frequency of mutations was <10(-10) for H. influenzae and M. catarrhalis for telithromycin, azithromycin and clarithromycin. Telithromycin induced mutations at a lower rate than azithromycin and clarithromycin in streptococcal strains., Conclusion: Telithromycin showed a very limited ability to select for resistance in respiratory pathogens compared with azithromycin and clarithromycin.

This study aimed to evaluate the bactericidal rates of levofloxacin, gatifloxacin, penicillin, meropenem and rokitamycin against seven isolates of Bacillus anthracis clinically isolated between 1960 and 1970. After determination of MIC and MBC, time-kill experiments were carried out. Antimicrobial activity was evaluated at concentrations equal to 1 x, 2 x, 4 x and 8 x MIC after 0, 3, 6, 12 and 24 h of incubation with the drugs. Bactericidal activity was defined as a decrease in bacterial count of at least 3 log10. All the isolates were susceptible to all the antibiotics, by considering the antistaphylococcal breakpoints. Levofloxacin was bactericidal at 1 x MIC after 24 h and at 4 x MIC after 12 h, and gatifloxacin was bactericidal at 2 x MIC after 24 h and at 8 x MIC after 12 h. Meropenem, rokitamycin and penicillin also showed bactericidal activity at concentrations of 4 x and 8 x MIC, respectively, but only after 24 h incubation; after the same time, meropenem and rokitamycin showed a more marked killing than penicillin at 2 x MIC.