BACKGROUND
escherichia coli is worldwide the most frequent pathogen isolated from uncomplicated urinary tract infections   and, in bacteremia of nosocomial or community origin, it represents about the  <dig> % and  <dig> % of aetiologies, respectively  <cit> .
also klebsiella spp., especially klebsiella pneumoniae, are involved in uncomplicated uti for 5% and represent  <dig> % of bacteremias, the mortality of nosocomial infections being more than twice that of community-acquired infection  <cit> .
fluoroquinolones  are potent antimicrobial agents used for the treatment of a wide variety of community- and nosocomial- infections.
however, increasing resistance to fq in e. coli isolated from community acquired uti has been recently reported, with up to 29% of women harbouring fq resistant e. coli, although fq resistance rates varied significantly according to sex, age, type of urinary infection and geographic region  <cit> .
moreover, infections due to extended-spectrum beta-lactamases  - producing enterobacteriaceae are an emerging problem in the community since an high proportion of these microorganisms have been isolated from urine samples of women with uncomplicated uti  <cit> .
ciprofloxacin use and esbl production have been shown to be significantly correlated in a study on k. pneumoniae  <cit> .
esbl-producing strains have been shown to be significantly more frequent among ciprofloxacin-resistant e. coli than among ciprofloxacin-susceptible e. coli strains  <cit> .
moreover, prior use of fqs, an indwelling urinary catheter, and an invasive procedure within  <dig> hr prior to bacteremia have been identified as independent risk factors for ciprofloxacin resistance in bloodstream infections due to esbl e. coli and klebsiella spp.
<cit> .
several studies have assessed the ability of fqs to select for resistance by subculturing bacteria at concentrations close to mics.
however, the antimicrobial concentrations used in these studies were quite different from those actually acquired at the site of infection  <cit> .
for these reasons, we have recently modified the methodologies used to assess in vitro the selection for resistance by testing antimicrobial concentrations reported to occur in vivo  <cit> .
the aim of the present study was to compare the ability of levofloxacin, ciprofloxacin and prulifloxacin to in vitro select for resistance in e. coli and klebsiella spp.
clinical isolates at peak  and trough  plasma concentrations.
RESULTS
susceptibility to fluoroquinolones
basal mics of e. coli strains ranged from  <dig>  mg/l to  <dig> mg/l, from  <dig>  mg/l to  <dig>  mg/l and from  <dig>  mg/l to  <dig>  mg/l for levofloxacin, ciprofloxacin and prulifloxacin, respectively.
mics of klebsiella spp.
ranged between  <dig>  mg/l and  <dig> mg/l,  <dig>  mg/l and  <dig>  mg/l, and  <dig>  and  <dig>  mg/l for levofloxacin, ciprofloxacin and prulifloxacin, respectively.
frequency of mutation
levofloxacin,  <dig> and  <dig> mg, and ciprofloxacin  <dig> mg limited bacterial growth with median frequencies of mutations below 10- <dig> at plasma cmax.
median frequencies of mutations for prulifloxacin were generally higher than comparators ranging from 10- <dig> to 10- <dig> and from 10- <dig> to 10- <dig> at plasma cmax in e. coli and klebsiella spp., respectively .
table  <dig> shows mic values of the strains that were able to grow in the presence of the above mentioned concentrations of all tested antimicrobials.
while no strain was able to grow at cmax for levofloxacin and ciprofloxacin,  <dig> and  <dig> strains grew at prulifloxacin cmax.
these strains showed increments in mics from  <dig> to  <dig> times for e. coli and from  <dig> to  <dig> times for klebsiella spp.
with respect to the basal values.
since in some instances, cmin for all the study drugs, except for levofloxacin at  <dig> mg dosage, were below mic values, some strains were able to diffusely grow on the agar plate.
for these strains, in order to detect any change in bacterial susceptibility, mics were evaluated for randomly sampled colonies .
lvx: levofloxacin; cip: ciprofloxacin; pru: prulifloxacin; cmax: peak plasma concentration; cmin: trough plasma concentration
* frequency of mutations was calculated only for strains with mic < cmin.
lvx: levofloxacin; cip: ciprofloxacin; pru: prulifloxacin; cmax: peak plasma concentration; cmin: trough plasma concentration
* mics were evaluated for all the tested strains
multi-step selection of resistant bacteria
lvx
lvx
cip
pru
lvx
lvx
cip
pru
lvx: levofloxacin; cip: ciprofloxacin; pru: prulifloxacin; pre-sel: mics before starting multi-step selection of resistance; i step: mics after the first passage on antibiotic gradient agar plates; v step: mics after the fifth passage on antibiotic gradient agar plates; x step: mics after the last passage on antibiotic gradient agar plates; x step free: mics after ten subcultures on antibiotic free agar plates.
after  <dig> passages on antibiotic gradient plates and  <dig> subcultures in antibiotic-free medium, the highest number of strains with mic higher than the resistance breakpoint was found for ciprofloxacin and prulifloxacin both in e. coli  and klebsiella spp.
.
only  <dig> strains with mic higher than resistance breakpoint were found with levofloxacin at  <dig> mg in e. coli and klebsiella spp., whereas no resistant strains selected with the  <dig> mg either in e. coli or in klebsiella spp.
.
characterization of acquired resistance
strains of e. coli that were selected by the multi-step assay and were able to maintain their resistance after  <dig> passages in antibiotic-free medium, were evaluated for acquired resistance.
among  <dig> resistant mutants, alterations in both gyra and parc were found in  <dig> mutants for ciprofloxacin  and prulifloxacin , while only alterations in gyra were found for levofloxacin.
as reported in table  <dig>  the  <dig> strains resistant to levofloxacin showed changes in ser83leu and asp87asn; while in ciprofloxacin- and prulifloxacin-resistant mutants, the mutations identified were ser83leu in gyra and ser80ile in parc.
the same mutations were not found in the respective parent strains.
lvx
cip
pru
discussion
wild-type e. coli and k. pneumoniae clinical isolates are susceptible to quinolones, but resistance to these agents in gram-negative bacteria has increased in recent years, probably caused by excessive and inappropriate use of these drugs  <cit> .
particularly, due to under-dosing and mono-therapy against moderately susceptible pathogens, fq resistance has developed among common pathogens, like e. coli and klebsiella spp., mainly conferred by esbls and ampc enzymes  <cit> .
esbl production has been reported to be two times more common in infected patients who received ciprofloxacin than in those who did not   <cit> .
in a study performed over  <dig> years in croatia on changes in susceptibility of e. coli from uti, moeal et al have shown a statistically significant change in antimicrobial resistance over that period only for ciprofloxacin  <cit> .
this has been hypothesized to be related to the inappropriate use of quinolones for humans as well as in veterinary medicine  <cit> .
prolonged use  of low dose  of the more potent fluoroquinolones such as ciprofloxacin or levofloxacin, has been shown to be the most significant risk factor for acquisition of resistance  <cit> .
strategies to counteract bacterial resistances include use of the appropriate dosages of these molecules for the correct indication and/or use of synergistic combinations, particularly in the more complicated infections.
results of this study indicate that levofloxacin presents the lowest frequencies of mutations at plasma cmax  and a lower propensity than prulifloxacin or ciprofloxacin to select in vitro for resistance.
in regard to genetic characterization of resistance, only alterations in gyra were found for levofloxacin, however, alterations in gyra and parc were found for ciprofloxacin and prulifloxacin.
point mutations within dna gyrase are known to cause a reduction in the affinity of the enzyme for fqs, decreasing the susceptibility of bacteria to these molecules.
topoisomerase iv is the second target for fq in the absence of susceptible gyrase.
therefore, multiple mutations in gyra and/or parc are required for high level fq resistance in e. coli  <cit> .
in our study, both ciprofloxacin and prulifloxacin resistant mutants presented mutations in gyra and parc, while levofloxacin resistance was found associated only with mutations in gyra.
these results seem to indicate that levofloxacin resistance at a concentration observed during treatment might develop more slowly and might be lower than resistance to the other fqs tested in the present study.
however, this study did not evaluated other mechanisms other than the target enzyme that might be involved in the observed resistant strains, including decreased intracellular drug accumulation as a result of alterations in the outer membrane proteins of the wall cell, or active efflux of the drug mediated by a number of efflux pumps.
as far as fq resistance in klebsiella spp.
is concerned, plasmid-mediated quinolone resistance mechanisms associated with the qnr gene and the aac-ib-cr gene in esbl producing strains have been described  <cit> .
the first encodes target protection proteins of the pent peptide repeat family and seems to be associated with low level quinolone resistance, while the aac-ib-cr gene encodes a variant of the common aminoglycoside acetyltransferase which is able to reduce the activity of some fq, thus enhancing the selection of chromosomal mutations  <cit> .
although in the present study the presence of plasmid-mediated resistance was not investigated, it can not be excluded that these genes might be involved in selection of resistance observed after serial exposure to fluoroquinolones.
in a previous study, we have shown that combinations of a fluoroquinolone with a beta-lactam may both provide improved antimicrobial activity and limit the occurrence of resistance in esbl-producing e. coli clinical isolates  <cit> .
therefore, the use of combination therapy could be an attractive strategy to limit occurrence of resistance.
CONCLUSIONS
in conclusion, among the tested fluoroquinolones, levofloxacin was the most able to limit occurrence of resistance in vitro.
however, in order to limit the occurrence of resistance, appropriate dosages of fluoroquinolones should be respected in the therapy of infections caused by enterobacteriaceae, as well as use of synergistic combinations in the most complicated infections.

 escherichia coli is worldwide the most frequent pathogen isolated from uncomplicated urinary tract infections and, in bacteremia of nosocomial or community origin, it represents about the % and % of aetiologies, respectively. also klebsiella spp., especially klebsiella pneumoniae, are involved in uncomplicated uti for 5% and represent % of bacteremias, the mortality of nosocomial infections being more than twice that of community-acquired infection. fluoroquinolones are potent antimicrobial agents used for the treatment of a wide variety of community- and nosocomial- infections. however, increasing resistance to fq in e. coli isolated from community acquired uti has been recently reported, with up to 29% of women harbouring fq resistant e. coli, although fq resistance rates varied significantly according to sex, age, type of urinary infection and geographic region. moreover, infections due to extended-spectrum beta-lactamases - producing enterobacteriaceae are an emerging problem in the community since an high proportion of these microorganisms have been isolated from urine samples of women with uncomplicated uti. ciprofloxacin use and esbl production have been shown to be significantly correlated in a study on k. pneumoniae. esbl-producing strains have been shown to be significantly more frequent among ciprofloxacin-resistant e. coli than among ciprofloxacin-susceptible e. coli strains. moreover, prior use of fqs, an indwelling urinary catheter, and an invasive procedure within hr prior to bacteremia have been identified as independent risk factors for ciprofloxacin resistance in bloodstream infections due to esbl e. coli and klebsiella spp.. several studies have assessed the ability of fqs to select for resistance by subculturing bacteria at concentrations close to mics. however, the antimicrobial concentrations used in these studies were quite different from those actually acquired at the site of infection. for these reasons, we have recently modified the methodologies used to assess in vitro the selection for resistance by testing antimicrobial concentrations reported to occur in vivo. the aim of the present study was to compare the ability of levofloxacin, ciprofloxacin and prulifloxacin to in vitro select for resistance in e. coli and klebsiella spp. clinical isolates at peak and trough plasma concentrations.  susceptibility to fluoroquinolones basal mics of e. coli strains ranged from mg/l to mg/l, from mg/l to mg/l and from mg/l to mg/l for levofloxacin, ciprofloxacin and prulifloxacin, respectively. mics of klebsiella spp. ranged between mg/l and mg/l, mg/l and mg/l, and and mg/l for levofloxacin, ciprofloxacin and prulifloxacin, respectively. frequency of mutation levofloxacin, and mg, and ciprofloxacin mg limited bacterial growth with median frequencies of mutations below 10- at plasma cmax. median frequencies of mutations for prulifloxacin were generally higher than comparators ranging from 10- to 10- and from 10- to 10- at plasma cmax in e. coli and klebsiella spp., respectively. table shows mic values of the strains that were able to grow in the presence of the above mentioned concentrations of all tested antimicrobials. while no strain was able to grow at cmax for levofloxacin and ciprofloxacin, and strains grew at prulifloxacin cmax. these strains showed increments in mics from to times for e. coli and from to times for klebsiella spp. with respect to the basal values. since in some instances, cmin for all the study drugs, except for levofloxacin at mg dosage, were below mic values, some strains were able to diffusely grow on the agar plate. for these strains, in order to detect any change in bacterial susceptibility, mics were evaluated for randomly sampled colonies. lvx: levofloxacin; cip: ciprofloxacin; pru: prulifloxacin; cmax: peak plasma concentration; cmin: trough plasma concentration * frequency of mutations was calculated only for strains with mic < cmin. lvx: levofloxacin; cip: ciprofloxacin; pru: prulifloxacin; cmax: peak plasma concentration; cmin: trough plasma concentration * mics were evaluated for all the tested strains multi-step selection of resistant bacteria lvx lvx cip pru lvx lvx cip pru lvx: levofloxacin; cip: ciprofloxacin; pru: prulifloxacin; pre-sel: mics before starting multi-step selection of resistance; i step: mics after the first passage on antibiotic gradient agar plates; v step: mics after the fifth passage on antibiotic gradient agar plates; x step: mics after the last passage on antibiotic gradient agar plates; x step free: mics after ten subcultures on antibiotic free agar plates. after passages on antibiotic gradient plates and subcultures in antibiotic-free medium, the highest number of strains with mic higher than the resistance breakpoint was found for ciprofloxacin and prulifloxacin both in e. coli and klebsiella spp.. only strains with mic higher than resistance breakpoint were found with levofloxacin at mg in e. coli and klebsiella spp., whereas no resistant strains selected with the mg either in e. coli or in klebsiella spp.. characterization of acquired resistance strains of e. coli that were selected by the multi-step assay and were able to maintain their resistance after passages in antibiotic-free medium, were evaluated for acquired resistance. among resistant mutants, alterations in both gyra and parc were found in mutants for ciprofloxacin and prulifloxacin, while only alterations in gyra were found for levofloxacin. as reported in table the strains resistant to levofloxacin showed changes in ser83leu and asp87asn; while in ciprofloxacin- and prulifloxacin-resistant mutants, the mutations identified were ser83leu in gyra and ser80ile in parc. the same mutations were not found in the respective parent strains. lvx cip pru discussion wild-type e. coli and k. pneumoniae clinical isolates are susceptible to quinolones, but resistance to these agents in gram-negative bacteria has increased in recent years, probably caused by excessive and inappropriate use of these drugs. particularly, due to under-dosing and mono-therapy against moderately susceptible pathogens, fq resistance has developed among common pathogens, like e. coli and klebsiella spp., mainly conferred by esbls and ampc enzymes. esbl production has been reported to be two times more common in infected patients who received ciprofloxacin than in those who did not. in a study performed over years in croatia on changes in susceptibility of e. coli from uti, moeal et al have shown a statistically significant change in antimicrobial resistance over that period only for ciprofloxacin. this has been hypothesized to be related to the inappropriate use of quinolones for humans as well as in veterinary medicine. prolonged use of low dose of the more potent fluoroquinolones such as ciprofloxacin or levofloxacin, has been shown to be the most significant risk factor for acquisition of resistance. strategies to counteract bacterial resistances include use of the appropriate dosages of these molecules for the correct indication and/or use of synergistic combinations, particularly in the more complicated infections. results of this study indicate that levofloxacin presents the lowest frequencies of mutations at plasma cmax and a lower propensity than prulifloxacin or ciprofloxacin to select in vitro for resistance. in regard to genetic characterization of resistance, only alterations in gyra were found for levofloxacin, however, alterations in gyra and parc were found for ciprofloxacin and prulifloxacin. point mutations within dna gyrase are known to cause a reduction in the affinity of the enzyme for fqs, decreasing the susceptibility of bacteria to these molecules. topoisomerase iv is the second target for fq in the absence of susceptible gyrase. therefore, multiple mutations in gyra and/or parc are required for high level fq resistance in e. coli. in our study, both ciprofloxacin and prulifloxacin resistant mutants presented mutations in gyra and parc, while levofloxacin resistance was found associated only with mutations in gyra. these results seem to indicate that levofloxacin resistance at a concentration observed during treatment might develop more slowly and might be lower than resistance to the other fqs tested in the present study. however, this study did not evaluated other mechanisms other than the target enzyme that might be involved in the observed resistant strains, including decreased intracellular drug accumulation as a result of alterations in the outer membrane proteins of the wall cell, or active efflux of the drug mediated by a number of efflux pumps. as far as fq resistance in klebsiella spp. is concerned, plasmid-mediated quinolone resistance mechanisms associated with the qnr gene and the aac-ib-cr gene in esbl producing strains have been described. the first encodes target protection proteins of the pent peptide repeat family and seems to be associated with low level quinolone resistance, while the aac-ib-cr gene encodes a variant of the common aminoglycoside acetyltransferase which is able to reduce the activity of some fq, thus enhancing the selection of chromosomal mutations. although in the present study the presence of plasmid-mediated resistance was not investigated, it can not be excluded that these genes might be involved in selection of resistance observed after serial exposure to fluoroquinolones. in a previous study, we have shown that combinations of a fluoroquinolone with a beta-lactam may both provide improved antimicrobial activity and limit the occurrence of resistance in esbl-producing e. coli clinical isolates. therefore, the use of combination therapy could be an attractive strategy to limit occurrence of resistance.  in conclusion, among the tested fluoroquinolones, levofloxacin was the most able to limit occurrence of resistance in vitro. however, in order to limit the occurrence of resistance, appropriate dosages of fluoroquinolones should be respected in the therapy of infections caused by enterobacteriaceae, as well as use of synergistic combinations in the most complicated infections. 
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. e. coli resistant strains selected after multi-step selection were characterized for dna mutations by sequencing gyra, gyrb, parc and pare genes. both ciprofloxacin- and prulifloxacin-resistant mutants presented mutations in gyra and parc, while levofloxacin resistance was found associated only to mutations in gyra. frequencies of mutations for levofloxacin and ciprofloxacin were less than 10- at peak concentration, while for prulifloxacin they ranged from <10- to 10- the lowest number of resistant mutants after multistep selection was selected by levofloxacin followed by ciprofloxacin and prulifloxacin. fluoroquinolones are potent antimicrobial agents used for the treatment of a wide variety of community- and nosocomial- infections. 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. multi-step selection of resistance was evaluated by performing serial cultures on agar plates containing a linear gradient from trough to peak antimicrobial concentrations, followed by subcultures on antibiotic-free agar.
 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 serial cultures on agar plates containing a linear gradient from trough to peak antimicrobial concentrations, followed by 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.  frequencies of mutations for levofloxacin and ciprofloxacin were less than 10- at peak concentration, while for prulifloxacin they ranged from <10- to 10- 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.  among the tested fluoroquinolones, levofloxacin was the most capable of limiting the occurrence of resistance. 