BACKGROUND
the development of bacterial resistance has resulted from a variety of factors, including drug overuse and drug misuse  <cit> , both in the environment and during therapy  <cit> .
even the commonly accepted treatment strategy of killing susceptible pathogens contributes to the problem by allowing selective amplification of resistant mutants during the treatment  <cit> .
according to a proposed hypothesis, resistant mutants selectively amplify at antibiotic concentrations within the mutant selection window , drug concentrations between the boundary of mic <dig>  and mutant prevention concentration   <cit> .
the mutant selection window hypothesis was initially proposed using agar plate assays  <cit> , and then explored in several in vitro or in vivo model  <cit> .
however, as far as we know, the time-dependent drugs such as cephalosporin were rarely reported.
cefquinome is a fourth generation broad-spectrum cephalosporin antibiotic, which was developed solely for veterinary use and approved for the treatment of respiratory tract disease, acute mastitis and footrot in cattle, calf septicaemia, respiratory diseases in pigs and metritis-mastitis-agalactia syndrome in sow  <cit> .
in order to reduce the occurring of cefquinome resistance and even the resistance gene transmission between herd and human, the study of mutant selection window is urgent which could be used as a framework for the design of dosage regimen of cefquinome therapy.
localized infection is particularly suitable for determination the mutant selection window hypothesis in in vivo  <cit> .
one feasible system is the tissue-cage infection model  <cit> , which used a tissue-cage with holes on its surface implanted surgically into subcutaneous tissue of an animal.
in piglets’ body, the surface of the cage becomes encapsulated by connective tissue 2- <dig> weeks after implantation, and the interior is filled with tissue-cage fluid.
bacterial cultures are injected into the cage and remained there until eliminated by host defenses and antimicrobial treatment.
in this study, above  <dig> cfu of e. coli atcc  <dig> were injected into the tissue-cages implanted in piglets, and various doses and intervals of cefquinome were administered intramuscularly.
the objective was to validate that the resistant mutants would be selected predominantly when drug concentrations maintained inside a concentration window, the boundaries of which determined by agar plate assays.
it is hoped that this model would have the capability to provide a clear demonstration of the mutant selection window in vivo and support arguments for how antimicrobial dosage regimens adjustment could severely restrict the amplification and enrichment of resistant mutant for cefquinome.
methods
antimicrobials and chemicals
penicillin, as a sodium salt for injection, and cefquinome for injection were purchased from hebei yuanzheng pharmaceutical co., ltd., p.r.
china.
cefquinome standard was from china institute of veterinary drugs control, beijing, p.r.
china.
acetonitrile and formic acid  were from fisher scientific.
mic <dig>  mic, and mpc determination
e. coli strain atcc  <dig> stored at -70°c was grown in mueller-hinton broth or on mueller-hinton agar.
mic <dig> and mpc were determined as described elsewhere  <cit> .
briefly, for mic <dig>  bacterial cultures were grown overnight  in the constant temperature oscillation incubator at 37°c, 220 r/min, normal atmosphere, serially diluted, and approximately  <dig> cells were applied to agar plates containing various concentrations of cefquinome.
after incubation at 37°c for 16-18 h, bacterial colonies were counted, and the fraction relative to the bacterial inoculum was calculated.
drug concentration that inhibited growth by 99% was defined as mic <dig>  for mpc, above  <dig> cells were applied to multiple cefquinome-containing agar plates.
after incubation at 37°c for a total of 96 h and the examination of the appearance of colonies every 24 h, mpc was recorded as the lowest antibiotic concentration at which no colonies grew on an agar plate.
the mic <dig> and mpc were determined in the five independent experiments.
tissue-cage infection model
healthy castrated cross-bred piglets ,weight ranging from  <dig> to 30 kg, were housed individually and fed antibiotic-free food twice a day.
water was available ad libitum.
the experimental protocol was approved by the committee on the ethics of animals of south china agricultural university .
the tissue-cages were made in-house from platinum-cured medical grade silicone tubing  and modified slightly from similar cages described by sidhu et al.
<cit> .
briefly, the dimensions of the tissue-cages were of 65 mm length, 18 mm external diameter and 13 mm internal diameter.
each cage had  <dig> identical holes and each hole has a surface area of  <dig>  mm2; the total exchange surface area was  <dig>  cm <dig>
two tissue-cages were implanted subcutaneously in each animal, one on either side of the neck approximately equidistant from the jugular vein and spinal cord under aseptic conditions.
surgical insertion was carried out under deep sedation  and local infiltration anaesthesia  in piglets.
after surgery, the piglets were treated with intramuscular penicillin  twice a day for 3- <dig> days to prevent infection.
the non-steroidal anti-inflammatory drug  was provided for analgesia in post-operation simultaneously.
by 4 weeks after implantation, each tissue-cage had become sealed with a thin layer of connective tissue and had been filled with clear, yellowish tissue-cage fluid.
above 1010 cfu of exponentially growing e. coli atcc  <dig> culture was concentrated in 1 ml of saline and injected into each tissue-cage.
two days after infection,  <dig>  ml of tissue-cage fluid was withdrawn from each cage for a viable-bacteria count.
piglets having above 108 cfu/ml viable bacterial cells in tissue-cage fluid were treated with various doses and intervals of cefquinome.
pharmacokinetic measurements
eighteen piglets were randomly allocated to  <dig> administration groups and treated at  <dig> ,  <dig> ,  <dig> ,  <dig> , or  <dig>  mg/kg of body weight once a day  or  <dig>  and  <dig>  mg/kg of body weight twice a day .
<dig> ,  <dig> ,  <dig>  mg/kg groups had  <dig> piglets and  <dig> tissue cages of each group.
<dig> ,  <dig>  mg/kg  groups had  <dig> piglets and  <dig> tissue cages of each group, which had one more piglet compared to  <dig> ,  <dig> ,  <dig>  mg/kg groups respectively because these two dosages easily induced resistant mutation.
and this series of dosages were determined by recommended dose which was 2 mg cefquinome/kg body weight in intramuscularly once daily for 3- <dig> days  <cit>  and pre-experiments data .
cefquinome were administrated intramuscularly  for consecutive  <dig> times beginning on the 3rd day after infection with e. coli atcc  <dig> for every piglet in administration groups.
the control group, three piglets, received sterile physiological saline  simultaneously in the same way.
tissue-cage fluid  was collected from the cage at  <dig>   <dig>   <dig>   <dig>   <dig>  and 24 h after each administration in group with 24 h interval.
for groups with 12 h interval, samples were collected at  <dig>   <dig>   <dig>   <dig>  12 h after each administration.
fluid samples were clarified by centrifugation at  <dig> 000 × g for 10 min and stored at -20°c.
the concentrations of cefquinome was determined using an agilent  <dig> series high performance liquid chromatography and an agilent  <dig> triple quadrupole mass spectrometer equipped with an electrospray ionization source .
the chromatographic separation was achieved on a phenonenex bds c <dig> column  at 40°c with a thermostat column oven .
the mobile phase consisted of solution a  and solution b  at  <dig>  ml/min flow rate.
the gradient elution was: 0-1 min, 5% b; 1- <dig>  min, 60% b;  <dig> -10 min, 5% b. the injection volume was 5 μl.
a calibrated curve was constructed by adding a known amount of cefquinome to blank tissue-cage fluid over concentrations ranged from  <dig>  μg/ml to 1 μg/ml.
the lower limit of quantification  of cefquinome was 5 ng/ml.
the recoveries of cefquinome in tissue-cage fluid were  <dig>  ±  <dig> % .
the coefficients of variability  were all < 10% for both intra-assay and inter-assay variation.
pharmacokinetic/pharmacodynamic  indices such as t>mic <dig>  t>mpc, auc/mic <dig>  auc/mpc, cmax/mic <dig>  cmax/mpc were calculated according to a noncompartmental analysis using winnonlin programme .
the liner trapezoidal rule was used to calculate the area under the concentration-time curve .
all pk/pd indices calculations referred to the 12 h and 24 h dosing interval immediately following the fifth injection after finishing administration using the cefquinome concentrations in tissue cage fluid.
loss of susceptibility to cefquinome
potential loss of susceptibility was monitored in tissue-cage fluid  obtained daily before and during the cefquinome treatment  and  <dig> and 48 h after the termination of treatment.
to amplify cultures, half of each sample was incubated overnight in drug-free mueller-hinton broth, and then the mic was determined with the clsi  <cit>  agar dilution method.
the other half of each sample was serially diluted with sterile physiological saline and applied to agar either lacking drug or containing cefquinome at 1 × mic of the starting culture.
after incubation at 37°c for 24-48 h, colonies were calculated, and the fraction of mutants in the population was calculated.
resistant mutants  were also chosen randomly from samples that had cefquinome concentrations predominantly in the lower, middle, or upper part of the selection window with 12 h or 24 h interval administration.
single colonies of these mutants were passaged  <dig> times on drug-free agar, and the mic to cefquinome was then determined.
statistical analysis
fisher’s exact test was used for statistical analysis of the pk/pd data, with an infected but untreated set of piglets  as a control.
p <  <dig>  was considered to be statistically significant.
RESULTS
bacteria count in tissue-cage model
two perforated tissue-cages were implanted into each piglet.
when above 1010 cfu bacteria were injected into an implanted cage, no severe illness or distress occurred during a 10-day observation.
bacterial concentrations remained constant at about 108 cfu/ml when piglets were treated intramuscularly for  <dig> times with sterile physiological saline once or twice daily.
administration of cefquinome at  <dig>  mg/kg at 24 h interval slightly reduced bacterial numbers compared to the control during the trial.
administration of cefquinome at  <dig>  and  <dig>  mg/kg at 24 h interval observably reduced bacterial numbers for the first  <dig> administrations, but bacterial growth was observed later during treatment and during post-treatment.
administration of cefquinome at  <dig>  and  <dig>  mg/kg at 24 h interval caused bacterial numbers to decrease throughout treatment and remain low during the growth recovery phase .figure  <dig>
effect of cefquinome dose on bacterial inhibition in the tissue-cage model.
tissue-cage implantation and escherichia coli atcc  <dig> infection were done as described in materials and methods.
three days after infection, various doses  of cefquinome were administered intramuscular once daily for 5 days .
bacterial colony-forming units in tissue-cage fluid was monitored at 24 h intervals beginning 1 day before the initiation of cefquinome in treatment and ending 2 days after the termination of cefquinome treatment.
cefquinome concentrations
the values of mic, mic <dig>  and mpc were  <dig>  μg/ml,  <dig>  μg/ml, and  <dig>  μg/ml in present study, respectively.
cefquinome concentrations, determined in samples of tissue-cage fluid collected at various time points over several days, are shown in figure  <dig> .
there were totally  <dig> tissue cages analyzed and another  <dig> tissue cages were excluded because of bacterial pollution.
and the cefquinome concentrations shown for each of the  <dig> groups were the means of the concentrations from all animals/tissue cage fluids selected for those groups.
the boundaries of the mutant selection window were determined to be  <dig>  μg/ml  and  <dig>  μg/ml  by agar plate assays, which were the average values determined in the five independent experiments.
there are  <dig> groups  to display the different classifications  of cefquinome concentrations based on the msw boundary.figure  <dig>
effect of actual cefquinome concentration on loss of susceptibility and mutant enrichment.
tissue-cage implantation and escherichia coli atcc  <dig> infection were done as described in meterials and methods.
piglets having above 1 × 108 cfu/ml e.coli in tissue-cage fluid 2 days after infection were treated with various intramuscular doses of cefquinome once daily and twice daily for  <dig> times beginning 3 days after infection.
the dosage, administration at hour or day  <dig> of the x-axis, protocol for each panel was as follows: a <dig>   <dig> received  <dig>  mg/kg; a <dig>   <dig> received  <dig>  mg/kg; a <dig>   <dig> received  <dig>  mg/kg and  <dig> received  <dig>  mg/kg; a <dig>   <dig> received  <dig>  mg/kg and  <dig> received  <dig>  mg/kg; a <dig>   <dig> received  <dig>  mg/kg; a <dig>   <dig> received  <dig>  mg/kg and  <dig> received  <dig>  mg/kg; a <dig>   <dig> received  <dig>  mg/kg and  <dig> received  <dig>  mg/kg; a <dig>   <dig> received  <dig>  mg/kg.
cefquinome concentration in tissue-cage fluid was monitored at the indicated times after the administration of each dose .
the boundaries of the mutant selection window  were determined with the e.coli inoculum by agar plate assays.
tissue-cage fluid was sampled for bacteria at 24 h and 12 h intervals for  <dig> times starting immediately before the administration of the first dose of cefquinome.
loss of susceptibility  was monitored as an increase in mic averaged for all piglets in the group.
the fraction of resistant mutants  in each group of piglets was determined daily as the number of colonies grown on cefquinome-containing agar  relative to the number that grew on drug-free agar.
mics and mutant fraction
samples of e. coli atcc  <dig> in tissue-cage fluid after treatment with various doses were examined for susceptibility to cefquinome.
increases in mics were readily observed  when cefquinome concentrations were partially or totally inside the selection window .
when cefquinome concentrations maintained either below the mic <dig>  or above the mpc , no mic increase, either during  or after therapy , was observed .
when cefquinome concentrations overlapped the lower window boundary  and the cmax was lower than the middle concentration  of the msw during treatment , decreased susceptibility  was detected in  <dig> of  <dig> cages and the mic increased to  <dig>  μg/ml at day  <dig> .
when cefquinome concentrations crossed the upper boundary of the window  and the cmin was higher than the middle concentration of the msw during treatment , loss of susceptibility was detected in  <dig> of  <dig> cages and the mic also increased to  <dig>  μg/ml at day  <dig> .
as drug concentrations were over the mic <dig> and t>mpc did not exceed 25% of interval, susceptibility decreased in  <dig> of  <dig> cages and the mic increased to  <dig>  μg/ml at day  <dig> , regardless of the mean concentration located into the lower  or upper  part of the selection window.
in addition, cefquinome susceptibility decreased in all  <dig> cages and the mic increased to  <dig>  μg/ml at day  <dig>  in the condition of 12 h interval with drug concentrations approximately the same with the condition of 24 h interval .
the detailed information is shown in table  <dig> table  <dig>
values of pk/pd parameters and change of mic in different cefquinome concentrations
groups
pk/pd parameters
interval
mic
cefquinome pk/pd values were determined from tissue cage fluids using total drug concentrations.
n is the animal number per group.
the fraction of mutants dramatically increased  when drug concentrations fell inside the selection window.
and there was no difference no matter whether the concentration partly or wholly dropped into the msw .
overall, these data have shown that the selection window boundaries determined by agar plate assays fit well with results obtained in vivo in piglets for cefquinome.
correlation of pk/pd indices with mutant enrichment and amplification
pk/pd indices, such as auc <dig> h/mic <dig>  and time above the mic <dig>  provide an empirical way to relate antimicrobial dose to favorable treatment effect for bactericidal agents.
relationships between pk/pd indices, determined as steady-state values after the fifth dose, and lost of susceptibility are shown in table  <dig>  for cephalosporin, t>mic <dig> is the index most commonly associated with restricting susceptible cell growth.
only  <dig> of  <dig> tissue-cages lost susceptibility when t>mic99≤6 h or t>mic99%≤25% .
t>mpc is probably the appropriate parameter for the upper boundary of the selection window.
only  <dig> of  <dig> tissue-cages was lost susceptibility when t>mpc≥12 h or t>mpc%≥50% .
loss of bacterial susceptibility occurred in  <dig> of  <dig> tissue-cages  when tmsw% was between 25% and 100%, with t>mpc%≤25% simultaneously .table  <dig>
correlation of pharmacokinetic/pharmacodynamics  parameters with selection of resistance
pk/pd index, value
a
fraction of tissue-cages with resistant bacteria
p
b
t > mic99
cmax/mic99
all pk/pd parameters were determined using total drug concentrations in tissue cage fluid.
total  <dig> tissue cages were analyzed and  <dig> tissue cages excluded because of bacterial pollution.
atine in the window  is not presented in the table because it fell into  <dig> categories.
bp values were calculated by fisher’s exact test, with a set of  <dig> infected but untreated piglets  used as a control.
high values indicate no difference with the control.
other pk/pd indices also showed statistically significant  correlations with the selection of resistance .
when auc <dig> h/mic <dig> and auc <dig> h/mpc were considered, the mutant selection window extended from  <dig>  h to  <dig>  h and from  <dig>  h to  <dig>  h, respectively.
in another example, the selection window extended from  <dig>  to  <dig>  and from  <dig>  to  <dig> , respectively, when maximum concentration cmax/mic <dig> and cmax/mpc were considered.
discussion
antimicrobial resistance becomes an increasingly serious problem that is likely to require attention at many levels  <cit> .
issues concerning dosing are addressed by the mutant selection window hypothesis  <cit> .
the fundamental difference between the traditional mic-based strategies and the mpc-based approach is that the former requires bacteria to acquire only  <dig> mutation for growth in the presence of drug, whereas the latter requires  <dig> or more  <cit> .
considering the boundaries of the selection window, it need to be predictable on the basis of data obtained by clinical microbiological laboratories.
previous work has shown that static agar plate values of mic <dig> and mpc of fluoroquinolones fit well with selection window boundaries obtained in in vitro models  <cit> .
the data in figure  <dig> demonstrate that agar plate determinations of mic <dig> and mpc fit well with the boundaries of the selection window seen in vivo at the site of infection for cefquinome.
in pk/pd model, the t>mic  and auc <dig> h/mic  can be used empirically to predict favorable effect when susceptible populations are considered  <cit> .
herein, the t>mic <dig>  or auc <dig> h/mic <dig>  can be used to define the lower boundary of the selection window in pk/pd combined with msw model.
for the upper boundary of the window, as the mpc is the mic of the least-susceptible single-step mutant, t>mpc or auc <dig> h/mpc probably is the appropriate parameter  <cit> .
an in vitro study of e. coli treated with ciprofloxacin also argued for the use of auc <dig> h/mpc.
other pk/pd indices—such as cmax/mic <dig>  cmax/mpc, and time in the window —also showed a statistically significant  correlation with the selection of resistance.
in present study, a correction is required when concentrations are high enough to kill the resistant .
keeping antimicrobial concentrations above the mpc >12 h  or auc <dig> h/mpc > <dig>  h is a straightforward way to restrict the acquisition of resistance in this study.
however, it is still complicated that what concentrations made the susceptible strains acquired resistance inside the window.
in previous investigation, concentrations at the center of the window were suitable for selecting a double mutant in an in vitro model  <cit> .
in present piglet/perforated-tissue-cage system, the drug concentration needed to be inside the window for ≥75% of the interval for enrichment mutants when those concentrations fluctuated above and below the mpc.
when the concentrations fluctuated above and below the mic <dig>  they needed to be inside the window for only 25% of the interval.
this difference probably results from:  more abundant preexisting resistant mutant subpopulations being able to survive and propagate near the bottom of the window;  the killing of some mutants when drug concentrations are close to the top of the window;  the organism have a better growth fitness at a low concentration than a high condition  <cit> .
the fraction of mutants can increase either by mutant amplification  or by mutant enrichment .
to distinguish these situation, we determined the absolute number both of total and resistant bacteria.
when drug concentrations were inside the selection window, the total population size decreased and then gradually increased.
however, the mutant numbers were initially constant, indicating that a fractional increase may result from preferential killing of susceptible cells.
after several times of administration, amplification of mutants was observed at last .
thus, the selection of cefquinome-resistant mutants in vivo probably arose from both mutant amplification  and mutant enrichment .figure  <dig>
effect of cefquinome exposure on recovery of total and resistant bacteria.
concentrations of total bacteria and resistant mutants were determined in aliquots of tissue-cage fluid obtained at the indicated time points after the initiation of treatment.
representative two examples  are shown for piglets in which the cefquinome concentration was inside the mutant selection window, as determined in figure  <dig>
beta-lactam antibiotics, including penicillins, cephalosporins and carbapenems, are widely used not only in human but also in vet clinic.
production of beta-lactamase is one of major resistance mechanisms in gram-negative bacteria, which mostly refers to extended-spectrum beta-lactamases  existing largely in microorganisms of the family enterobacteriaceae  <cit> .
it was complicated to confirm the mechanism of acquisition resistant genes for sensitive strains exposure of antimicrobial agents, such as chromosome gene mutant and plasmid acquisition.
the present data could not display how the resistant property was produced by e. coli atcc  <dig>  more molecular technique, the pcr of target gene, the pfge of original strain and mutants, and plasmid profiles analysis at least, should be applied to determine the mutant gene which caused bacterial resistance.
those are the mainly study purposes of the next task in laboratory animals.
CONCLUSIONS
in conclusion, the data in figure  <dig> demonstrate that the mutant selection window agar plate determinations fit well with the piglets infected with e. coli atcc  <dig> of cefquinome treatment in vivo.
because agar plate assays are routine in clinical laboratories, implementation of selection window dosing strategy is feasible.
the next steps for the tissue-cage model are to obtain more data to confirm the boundary of msw.
for example, more virulent strains should be used to allow bacterial populations to reach  <dig> cells by in vivo growth from a smaller inoculum in further study.
abbreviations
mswmutant selection window
e.coliescherichia coli
micminimum inhibitory concentration
mpcmutant prevention concentration
pkpharmacokinetics
pdpharmacodynamics
aucarea under the curve
cfucolony forming unit
atccamerican type culture collection
nsaidnon-steroidal anti-inflammatory drug
hplchigh performance liquid chromatography
msmass spectrometry
lloqlower limit of quantification
cvcoefficients of variability
clsiclinical and laboratory standards institute
esblsextended-spectrum beta-lactamases
pcrpolymerase chain reaction
pfgepulsed field gel electrophoresis
competing interests
the authors declare that they have no competing interests.
authors’ contributions
designed and conducted the experiment: hd; bz.
animal management, health and welfare: xg; yl; xl.
collected and analysis samples: bz; mg; nz; xs.
analysis data: bz.
drafted the manuscript: bz.
all authors read and approved the final manuscript.

 the development of bacterial resistance has resulted from a variety of factors, including drug overuse and drug misuse, both in the environment and during therapy. even the commonly accepted treatment strategy of killing susceptible pathogens contributes to the problem by allowing selective amplification of resistant mutants during the treatment. according to a proposed hypothesis, resistant mutants selectively amplify at antibiotic concentrations within the mutant selection window, drug concentrations between the boundary of mic and mutant prevention concentration. the mutant selection window hypothesis was initially proposed using agar plate assays, and then explored in several in vitro or in vivo model. however, as far as we know, the time-dependent drugs such as cephalosporin were rarely reported. cefquinome is a fourth generation broad-spectrum cephalosporin antibiotic, which was developed solely for veterinary use and approved for the treatment of respiratory tract disease, acute mastitis and footrot in cattle, calf septicaemia, respiratory diseases in pigs and metritis-mastitis-agalactia syndrome in sow. in order to reduce the occurring of cefquinome resistance and even the resistance gene transmission between herd and human, the study of mutant selection window is urgent which could be used as a framework for the design of dosage regimen of cefquinome therapy. localized infection is particularly suitable for determination the mutant selection window hypothesis in in vivo. one feasible system is the tissue-cage infection model, which used a tissue-cage with holes on its surface implanted surgically into subcutaneous tissue of an animal. in piglets’ body, the surface of the cage becomes encapsulated by connective tissue 2- weeks after implantation, and the interior is filled with tissue-cage fluid. bacterial cultures are injected into the cage and remained there until eliminated by host defenses and antimicrobial treatment. in this study, above cfu of e. coli atcc were injected into the tissue-cages implanted in piglets, and various doses and intervals of cefquinome were administered intramuscularly. the objective was to validate that the resistant mutants would be selected predominantly when drug concentrations maintained inside a concentration window, the boundaries of which determined by agar plate assays. it is hoped that this model would have the capability to provide a clear demonstration of the mutant selection window in vivo and support arguments for how antimicrobial dosage regimens adjustment could severely restrict the amplification and enrichment of resistant mutant for cefquinome. methods antimicrobials and chemicals penicillin, as a sodium salt for injection, and cefquinome for injection were purchased from hebei yuanzheng pharmaceutical co., ltd., p.r. china. cefquinome standard was from china institute of veterinary drugs control, beijing, p.r. china. acetonitrile and formic acid were from fisher scientific. mic mic, and mpc determination e. coli strain atcc stored at -70°c was grown in mueller-hinton broth or on mueller-hinton agar. mic and mpc were determined as described elsewhere. briefly, for mic bacterial cultures were grown overnight in the constant temperature oscillation incubator at 37°c, 220 r/min, normal atmosphere, serially diluted, and approximately cells were applied to agar plates containing various concentrations of cefquinome. after incubation at 37°c for 16-18 h, bacterial colonies were counted, and the fraction relative to the bacterial inoculum was calculated. drug concentration that inhibited growth by 99% was defined as mic for mpc, above cells were applied to multiple cefquinome-containing agar plates. after incubation at 37°c for a total of 96 h and the examination of the appearance of colonies every 24 h, mpc was recorded as the lowest antibiotic concentration at which no colonies grew on an agar plate. the mic and mpc were determined in the five independent experiments. tissue-cage infection model healthy castrated cross-bred piglets,weight ranging from to 30 kg, were housed individually and fed antibiotic-free food twice a day. water was available ad libitum. the experimental protocol was approved by the committee on the ethics of animals of south china agricultural university. the tissue-cages were made in-house from platinum-cured medical grade silicone tubing and modified slightly from similar cages described by sidhu et al.. briefly, the dimensions of the tissue-cages were of 65 mm length, 18 mm external diameter and 13 mm internal diameter. each cage had identical holes and each hole has a surface area of  mm2; the total exchange surface area was  cm two tissue-cages were implanted subcutaneously in each animal, one on either side of the neck approximately equidistant from the jugular vein and spinal cord under aseptic conditions. surgical insertion was carried out under deep sedation and local infiltration anaesthesia in piglets. after surgery, the piglets were treated with intramuscular penicillin twice a day for 3- days to prevent infection. the non-steroidal anti-inflammatory drug was provided for analgesia in post-operation simultaneously. by 4 weeks after implantation, each tissue-cage had become sealed with a thin layer of connective tissue and had been filled with clear, yellowish tissue-cage fluid. above 1010 cfu of exponentially growing e. coli atcc culture was concentrated in 1 ml of saline and injected into each tissue-cage. two days after infection,  ml of tissue-cage fluid was withdrawn from each cage for a viable-bacteria count. piglets having above 108 cfu/ml viable bacterial cells in tissue-cage fluid were treated with various doses and intervals of cefquinome. pharmacokinetic measurements eighteen piglets were randomly allocated to administration groups and treated at,,,, or  mg/kg of body weight once a day or and  mg/kg of body weight twice a day.,,  mg/kg groups had piglets and tissue cages of each group.,  mg/kg groups had piglets and tissue cages of each group, which had one more piglet compared to,,  mg/kg groups respectively because these two dosages easily induced resistant mutation. and this series of dosages were determined by recommended dose which was 2 mg cefquinome/kg body weight in intramuscularly once daily for 3- days and pre-experiments data. cefquinome were administrated intramuscularly for consecutive times beginning on the 3rd day after infection with e. coli atcc for every piglet in administration groups. the control group, three piglets, received sterile physiological saline simultaneously in the same way. tissue-cage fluid was collected from the cage at and 24 h after each administration in group with 24 h interval. for groups with 12 h interval, samples were collected at 12 h after each administration. fluid samples were clarified by centrifugation at 000 × g for 10 min and stored at -20°c. the concentrations of cefquinome was determined using an agilent series high performance liquid chromatography and an agilent triple quadrupole mass spectrometer equipped with an electrospray ionization source. the chromatographic separation was achieved on a phenonenex bds c column at 40°c with a thermostat column oven. the mobile phase consisted of solution a and solution b at  ml/min flow rate. the gradient elution was: 0-1 min, 5% b; 1-  min, 60% b; -10 min, 5% b. the injection volume was 5 μl. a calibrated curve was constructed by adding a known amount of cefquinome to blank tissue-cage fluid over concentrations ranged from  μg/ml to 1 μg/ml. the lower limit of quantification of cefquinome was 5 ng/ml. the recoveries of cefquinome in tissue-cage fluid were  ±  %. the coefficients of variability were all < 10% for both intra-assay and inter-assay variation. pharmacokinetic/pharmacodynamic indices such as t>mic t>mpc, auc/mic auc/mpc, cmax/mic cmax/mpc were calculated according to a noncompartmental analysis using winnonlin programme. the liner trapezoidal rule was used to calculate the area under the concentration-time curve. all pk/pd indices calculations referred to the 12 h and 24 h dosing interval immediately following the fifth injection after finishing administration using the cefquinome concentrations in tissue cage fluid. loss of susceptibility to cefquinome potential loss of susceptibility was monitored in tissue-cage fluid obtained daily before and during the cefquinome treatment and and 48 h after the termination of treatment. to amplify cultures, half of each sample was incubated overnight in drug-free mueller-hinton broth, and then the mic was determined with the clsi agar dilution method. the other half of each sample was serially diluted with sterile physiological saline and applied to agar either lacking drug or containing cefquinome at 1 × mic of the starting culture. after incubation at 37°c for 24-48 h, colonies were calculated, and the fraction of mutants in the population was calculated. resistant mutants were also chosen randomly from samples that had cefquinome concentrations predominantly in the lower, middle, or upper part of the selection window with 12 h or 24 h interval administration. single colonies of these mutants were passaged times on drug-free agar, and the mic to cefquinome was then determined. statistical analysis fisher’s exact test was used for statistical analysis of the pk/pd data, with an infected but untreated set of piglets as a control. p <  was considered to be statistically significant.  bacteria count in tissue-cage model two perforated tissue-cages were implanted into each piglet. when above 1010 cfu bacteria were injected into an implanted cage, no severe illness or distress occurred during a 10-day observation. bacterial concentrations remained constant at about 108 cfu/ml when piglets were treated intramuscularly for times with sterile physiological saline once or twice daily. administration of cefquinome at  mg/kg at 24 h interval slightly reduced bacterial numbers compared to the control during the trial. administration of cefquinome at and  mg/kg at 24 h interval observably reduced bacterial numbers for the first administrations, but bacterial growth was observed later during treatment and during post-treatment. administration of cefquinome at and  mg/kg at 24 h interval caused bacterial numbers to decrease throughout treatment and remain low during the growth recovery phase.figure effect of cefquinome dose on bacterial inhibition in the tissue-cage model. tissue-cage implantation and escherichia coli atcc infection were done as described in materials and methods. three days after infection, various doses of cefquinome were administered intramuscular once daily for 5 days. bacterial colony-forming units in tissue-cage fluid was monitored at 24 h intervals beginning 1 day before the initiation of cefquinome in treatment and ending 2 days after the termination of cefquinome treatment. cefquinome concentrations the values of mic, mic and mpc were  μg/ml,  μg/ml, and  μg/ml in present study, respectively. cefquinome concentrations, determined in samples of tissue-cage fluid collected at various time points over several days, are shown in figure . there were totally tissue cages analyzed and another tissue cages were excluded because of bacterial pollution. and the cefquinome concentrations shown for each of the groups were the means of the concentrations from all animals/tissue cage fluids selected for those groups. the boundaries of the mutant selection window were determined to be  μg/ml and  μg/ml by agar plate assays, which were the average values determined in the five independent experiments. there are groups to display the different classifications of cefquinome concentrations based on the msw boundary.figure effect of actual cefquinome concentration on loss of susceptibility and mutant enrichment. tissue-cage implantation and escherichia coli atcc infection were done as described in meterials and methods. piglets having above 1 × 108 cfu/ml e.coli in tissue-cage fluid 2 days after infection were treated with various intramuscular doses of cefquinome once daily and twice daily for times beginning 3 days after infection. the dosage, administration at hour or day of the x-axis, protocol for each panel was as follows: a received  mg/kg; a received  mg/kg; a received  mg/kg and received  mg/kg; a received  mg/kg and received  mg/kg; a received  mg/kg; a received  mg/kg and received  mg/kg; a received  mg/kg and received  mg/kg; a received  mg/kg. cefquinome concentration in tissue-cage fluid was monitored at the indicated times after the administration of each dose. the boundaries of the mutant selection window were determined with the e.coli inoculum by agar plate assays. tissue-cage fluid was sampled for bacteria at 24 h and 12 h intervals for times starting immediately before the administration of the first dose of cefquinome. loss of susceptibility was monitored as an increase in mic averaged for all piglets in the group. the fraction of resistant mutants in each group of piglets was determined daily as the number of colonies grown on cefquinome-containing agar relative to the number that grew on drug-free agar. mics and mutant fraction samples of e. coli atcc in tissue-cage fluid after treatment with various doses were examined for susceptibility to cefquinome. increases in mics were readily observed when cefquinome concentrations were partially or totally inside the selection window. when cefquinome concentrations maintained either below the mic or above the mpc, no mic increase, either during or after therapy, was observed. when cefquinome concentrations overlapped the lower window boundary and the cmax was lower than the middle concentration of the msw during treatment, decreased susceptibility was detected in of cages and the mic increased to  μg/ml at day. when cefquinome concentrations crossed the upper boundary of the window and the cmin was higher than the middle concentration of the msw during treatment, loss of susceptibility was detected in of cages and the mic also increased to  μg/ml at day. as drug concentrations were over the mic and t>mpc did not exceed 25% of interval, susceptibility decreased in of cages and the mic increased to  μg/ml at day, regardless of the mean concentration located into the lower or upper part of the selection window. in addition, cefquinome susceptibility decreased in all cages and the mic increased to  μg/ml at day in the condition of 12 h interval with drug concentrations approximately the same with the condition of 24 h interval. the detailed information is shown in table  table values of pk/pd parameters and change of mic in different cefquinome concentrations groups pk/pd parameters interval mic cefquinome pk/pd values were determined from tissue cage fluids using total drug concentrations. n is the animal number per group. the fraction of mutants dramatically increased when drug concentrations fell inside the selection window. and there was no difference no matter whether the concentration partly or wholly dropped into the msw. overall, these data have shown that the selection window boundaries determined by agar plate assays fit well with results obtained in vivo in piglets for cefquinome. correlation of pk/pd indices with mutant enrichment and amplification pk/pd indices, such as auc h/mic and time above the mic provide an empirical way to relate antimicrobial dose to favorable treatment effect for bactericidal agents. relationships between pk/pd indices, determined as steady-state values after the fifth dose, and lost of susceptibility are shown in table  for cephalosporin, t>mic is the index most commonly associated with restricting susceptible cell growth. only of tissue-cages lost susceptibility when t>mic99≤6 h or t>mic99%≤25%. t>mpc is probably the appropriate parameter for the upper boundary of the selection window. only of tissue-cages was lost susceptibility when t>mpc≥12 h or t>mpc%≥50%. loss of bacterial susceptibility occurred in of tissue-cages when tmsw% was between 25% and 100%, with t>mpc%≤25% simultaneously.table correlation of pharmacokinetic/pharmacodynamics parameters with selection of resistance pk/pd index, value a fraction of tissue-cages with resistant bacteria p b t > mic99 cmax/mic99 all pk/pd parameters were determined using total drug concentrations in tissue cage fluid. total tissue cages were analyzed and tissue cages excluded because of bacterial pollution. atine in the window is not presented in the table because it fell into categories. bp values were calculated by fisher’s exact test, with a set of infected but untreated piglets used as a control. high values indicate no difference with the control. other pk/pd indices also showed statistically significant correlations with the selection of resistance. when auc h/mic and auc h/mpc were considered, the mutant selection window extended from  h to  h and from  h to  h, respectively. in another example, the selection window extended from to and from to, respectively, when maximum concentration cmax/mic and cmax/mpc were considered. discussion antimicrobial resistance becomes an increasingly serious problem that is likely to require attention at many levels. issues concerning dosing are addressed by the mutant selection window hypothesis. the fundamental difference between the traditional mic-based strategies and the mpc-based approach is that the former requires bacteria to acquire only mutation for growth in the presence of drug, whereas the latter requires or more. considering the boundaries of the selection window, it need to be predictable on the basis of data obtained by clinical microbiological laboratories. previous work has shown that static agar plate values of mic and mpc of fluoroquinolones fit well with selection window boundaries obtained in in vitro models. the data in figure  demonstrate that agar plate determinations of mic and mpc fit well with the boundaries of the selection window seen in vivo at the site of infection for cefquinome. in pk/pd model, the t>mic and auc h/mic can be used empirically to predict favorable effect when susceptible populations are considered. herein, the t>mic or auc h/mic can be used to define the lower boundary of the selection window in pk/pd combined with msw model. for the upper boundary of the window, as the mpc is the mic of the least-susceptible single-step mutant, t>mpc or auc h/mpc probably is the appropriate parameter. an in vitro study of e. coli treated with ciprofloxacin also argued for the use of auc h/mpc. other pk/pd indices—such as cmax/mic cmax/mpc, and time in the window —also showed a statistically significant correlation with the selection of resistance. in present study, a correction is required when concentrations are high enough to kill the resistant. keeping antimicrobial concentrations above the mpc >12 h or auc h/mpc >  h is a straightforward way to restrict the acquisition of resistance in this study. however, it is still complicated that what concentrations made the susceptible strains acquired resistance inside the window. in previous investigation, concentrations at the center of the window were suitable for selecting a double mutant in an in vitro model. in present piglet/perforated-tissue-cage system, the drug concentration needed to be inside the window for ≥75% of the interval for enrichment mutants when those concentrations fluctuated above and below the mpc. when the concentrations fluctuated above and below the mic they needed to be inside the window for only 25% of the interval. this difference probably results from: more abundant preexisting resistant mutant subpopulations being able to survive and propagate near the bottom of the window; the killing of some mutants when drug concentrations are close to the top of the window; the organism have a better growth fitness at a low concentration than a high condition. the fraction of mutants can increase either by mutant amplification or by mutant enrichment. to distinguish these situation, we determined the absolute number both of total and resistant bacteria. when drug concentrations were inside the selection window, the total population size decreased and then gradually increased. however, the mutant numbers were initially constant, indicating that a fractional increase may result from preferential killing of susceptible cells. after several times of administration, amplification of mutants was observed at last. thus, the selection of cefquinome-resistant mutants in vivo probably arose from both mutant amplification and mutant enrichment.figure effect of cefquinome exposure on recovery of total and resistant bacteria. concentrations of total bacteria and resistant mutants were determined in aliquots of tissue-cage fluid obtained at the indicated time points after the initiation of treatment. representative two examples are shown for piglets in which the cefquinome concentration was inside the mutant selection window, as determined in figure  beta-lactam antibiotics, including penicillins, cephalosporins and carbapenems, are widely used not only in human but also in vet clinic. production of beta-lactamase is one of major resistance mechanisms in gram-negative bacteria, which mostly refers to extended-spectrum beta-lactamases existing largely in microorganisms of the family enterobacteriaceae. it was complicated to confirm the mechanism of acquisition resistant genes for sensitive strains exposure of antimicrobial agents, such as chromosome gene mutant and plasmid acquisition. the present data could not display how the resistant property was produced by e. coli atcc more molecular technique, the pcr of target gene, the pfge of original strain and mutants, and plasmid profiles analysis at least, should be applied to determine the mutant gene which caused bacterial resistance. those are the mainly study purposes of the next task in laboratory animals.  in conclusion, the data in figure  demonstrate that the mutant selection window agar plate determinations fit well with the piglets infected with e. coli atcc of cefquinome treatment in vivo. because agar plate assays are routine in clinical laboratories, implementation of selection window dosing strategy is feasible. the next steps for the tissue-cage model are to obtain more data to confirm the boundary of msw. for example, more virulent strains should be used to allow bacterial populations to reach cells by in vivo growth from a smaller inoculum in further study. abbreviations mswmutant selection window e.coliescherichia coli micminimum inhibitory concentration mpcmutant prevention concentration pkpharmacokinetics pdpharmacodynamics aucarea under the curve cfucolony forming unit atccamerican type culture collection nsaidnon-steroidal anti-inflammatory drug hplchigh performance liquid chromatography msmass spectrometry lloqlower limit of quantification cvcoefficients of variability clsiclinical and laboratory standards institute esblsextended-spectrum beta-lactamases pcrpolymerase chain reaction pfgepulsed field gel electrophoresis competing interests the authors declare that they have no competing interests. authors’ contributions designed and conducted the experiment: hd; bz. animal management, health and welfare: xg; yl; xl. collected and analysis samples: bz; mg; nz; xs. analysis data: bz. drafted the manuscript: bz. all authors read and approved the final manuscript. 
maintenance of antimicrobial concentrations above the mpc for > 50% of administration interval is a straightforward way to restrict the acquisition of resistance in this tissue cage model. in order to inhibit the bacterial resistance production, the mutant selection window hypothesis with escherichia coli atcc exposed to cefquinome in an animal tissue-cage model was investigated. when auc h/mic and auc h/mpc were considered, the mutant selection window extended from  h to  h and from  h to  h, respectively. tissue-cage modelsmutant selection windowcefquinomepigletsissue-copyright-statement© the author 2014 e. coli lost susceptibility when drug concentrations fluctuated between the lower and upper boundaries of the window, which defined in vitro as the mic and the mpc respectively. localized infection with e. coli was established in piglets, and the infected animals were administrated intramuscularly with various doses and intervals of cefquinome to provide antibiotic concentrations below the mic between the mic and the mutant prevention concentration, and above the mpc. for pk/pd parameters, there were no mutant selection enrichment when t>mic was ≤ 25% or t>mpc was ≥ 50% of administration interval. this situation was achieved with daily intramuscular doses of 1 mg cefquinome/kg body weight.
 the resistance of cephalosporins is significantly serious in veterinary clinic. in order to inhibit the bacterial resistance production, the mutant selection window hypothesis with escherichia coli atcc exposed to cefquinome in an animal tissue-cage model was investigated.  localized infection with e. coli was established in piglets, and the infected animals were administrated intramuscularly with various doses and intervals of cefquinome to provide antibiotic concentrations below the mic between the mic and the mutant prevention concentration, and above the mpc. e. coli lost susceptibility when drug concentrations fluctuated between the lower and upper boundaries of the window, which defined in vitro as the mic and the mpc respectively. for pk/pd parameters, there were no mutant selection enrichment when t>mic was ≤ 25% or t>mpc was ≥ 50% of administration interval. when t>mic was > 25% and t>mpc was <50% of administration interval, resistance selection was observed. when auc h/mic and auc h/mpc were considered, the mutant selection window extended from  h to  h and from  h to  h, respectively.  these findings demonstrate that the msw exists in vivo for time-dependent antimicrobial agents, and its boundaries fit well with those determined in vitro. maintenance of antimicrobial concentrations above the mpc for > 50% of administration interval is a straightforward way to restrict the acquisition of resistance in this tissue cage model. this situation was achieved with daily intramuscular doses of 1 mg cefquinome/kg body weight. keywords tissue-cage modelsmutant selection windowcefquinomepigletsissue-copyright-statement© the author 2014 