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187（ 1 ）Relation of %T＞MIC of piperacillin to
the clinical outcome in the treatment ofGram-negative bacterial infections
Ryota Okuma1,*, Yukihiro Hamada2, Harue Kohara1,
Takeshi Hirayama3,4, Yusuke Kumamoto5
and Masakazu Kuroyama1,4
12

Department of Pharmacy, Kitasato University East Hospital
Department of Pharmacy, Tokyo Womenʼs Medical University Hospital3
Department of Pharmacy, Kitasato University Hospital
4
Kitasato University School of Pharmacy
5
Kitasato University Department of Surgery(Received for publication March 27, 2017)

There are few reports of direct assessment of the percent duration of the dosinginterval for which the concentration of the antibiotic in the blood remains above theminimum inhibitory concentration (MIC) (time above the MIC% [%fT＞MIC]) andthe clinical outcome for piperacillin (PIPC). Therefore, we investigated therelationship between the %fT＞MIC and the outcomes in patients receiving PIPC
therapy. In patients treated with PIPC at Kitasato University East Hospital, the %fT＞MIC for the antibiotic was determined retrospectively for each patient from theserum concentrations of the drug plotted over time, and the relationship between the%fT＞MIC and the therapeutic response rate was calculated by logistic regression
analysis. Evaluation of the efﬁcacy of the drug was carried out on the basis of its
bacteriological effects (elimination of the bacterial pathogen). The analysis revealed
that a response rate of ＞90% was achieved when the %fT＞MIC was ≥60%.
Assessment of the relationship between the emergence of resistant organisms and the%fT＞MIC in the non-responders revealed that the emergence of resistant organisms
can be prevented if a %fT＞MIC of 60% can be achieved, at which the drug is
known to exert maximal bactericidal effect. In conclusion, this study suggested thatmaintaining the %fT＞MIC at a target of 60% in the dosage design of PIPC therapy
would result in improved clinical outcomes.

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1. Introduction
Since the 1990s, rapid progress has been made in research on the pharmacokinetics (PK) andpharmacodynamics (PD) of antimicrobial agents, which has contributed in great measure to appropriate treatment of infections and development and marketing of novel antimicrobial agents.
Proper use antimicrobial drugs on the basis of the patient background characteristics, causative
organisms, site of infection, and the PK/PD characteristics of the drugs is recommended in the
Antimicrobial Stewardship Guideline released jointly by the Infection Disease Society of America (IDSA) and the Society for Healthcare Epidemiology of America (SHEA)1). It has become
possible to gain a better understanding of the pharmacologic effects in relation to the changes ofthe serum drug concentrations over time by PK/PD analysis, and the information secured thereby
is particularly important for proper use of antimicrobial agents2–4).
Among the most useful outcomes of studies on the PK/PD is clariﬁcation of the PK/PD parameters that are correlated with the bactericidal effects of antimicrobial agents in vivo. The PK/
PD parameters vary with the type of antimicrobial agent studied. In the case of β-lactam antibiotics, for example, it has been shown that the percent time over which the drug concentration remains over the minimum inhibitory concentration (MIC) relative to the dose interval, i.e., thetime above MIC% (%T＞MIC), represents a PK/PD parameter5). It has also been reported that in
the case of penicillin antibiotics, a bacterial growth-inhibitory effect can be achieved at doses
yielding a free-form (unbound) drug %T＞MIC (%fT＞MIC) of ≥30% and a maximum bactericidal effect can be obtained at doses yielding a %fT＞MIC of ≥50%6). It is of importance, therefore, to set the dosage and administration schedule so as to achieve an appropriate %fT＞MIC inpenicillin pharmacotherapy.
We previously assessed the dosage and administration schedule of tazobactam/piperacillin(TAZ/PIPC) from the viewpoint of PK/PD, by determining the association of the dosage and administration schedule with the %fT＞MIC7). The %fT＞MIC was calculated from the serum drugconcentrations, although the drug concentrations in the infected tissues also appear to have abearing on the actual therapeutic responses. Consequently, there is the possibility, if the %fT＞
MIC calculated from the serum concentrations of the drug is set as an indicator of the efﬁcacythat the drug levels in the infected tissues do not reach the MIC, and it is necessary to relate thetherapeutic responses to the %fT＞MIC when this parameter is applied in the clinical practice setting. In Japan, the currently approved daily dose levels of antimicrobial agents are frequently
lower than those in Europe and the United States; therefore, there are instances in which the
breakpoint stated in the guidelines of the Clinical and Laboratory Standards Institute (CLSI)8) andEuropean Committee on Antimicrobial Susceptibility Testing (EUCAST)9) cannot be directly utilized. The approved maximum dose of PIPC, a penicillin antibiotic, is 8.0 g/day in Japan. As of
March 2015, a rise in the maximum dose of this antibiotic has been approved and it is now feasi-

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189（ 3 ）ble in Japan to carry out treatment with this drug at the same dose levels as those in Europe andthe United States. However, there are no reports yet of direct assessment of the therapeutic responses to PIPC in relation to its %fT＞MIC.
Meanwhile, inappropriate antimicrobial drug administration that fails to provide sufﬁcientserum drug concentrations may give rise to not only failure of treatment, but also to diminution of
the antimicrobial susceptibility (emergence of resistant organisms)10,11). Treatment with an antimicrobial agent at its MIC, which is the lowest concentration at which it exerts its antimicrobial
effect, does not necessarily eliminate all susceptible organisms, and exposure to the drug at a concentration only slightly higher than the MIC may result in a selection of resistant organisms only.
The drug concentration at which the selection of resistant organisms is suppressed is termed mutant prevention concentration (MPC), and the concentration range between the MIC, at which resistant variants may be selected, and the MPC is referred to as the mutant selection window(MSW)12). There is, in our view, the need for a new parameter to determine the concentration atwhich emergence of resistant organisms would be suppressed, because it is almost impracticable
to determine the MPC in the clinical setting. The mortalities associated with hospital-acquired
pneumonia and ventilator-associated pneumonia are still high13), and one report has documented
the emergence of resistant organisms in approximately 10–50% of cases of hospital-acquired
pneumonia treated with β-lactam antimicrobial agents14). It has been suggested that in an in vitro
infection setting, the ratio of the trough concentration to the MIC (trough/MIC) of a β-lactam antimicrobial agent was associated with suppression of the emergence of resistant organisms15), although the relationship between the extent of exposure and the emergence of resistant organisms
still remains to be clariﬁed.
Under these circumstances, we conducted this study to assess the relationship between the%fT＞MIC and the clinical efﬁcacy and between the %fT＞MIC and emergence of drug-resistantorganisms by means of PK/PD analysis in patients receiving treatment with PIPC.

2. Patients and methods
2-1. Patients
From among inpatients treated with PIPC between January 2006 and March 2014 at theKitasato University East Hospital (this hospital, hereafter), those with bacterial isolates for which
the MICs of PIPC were determined at the time of bacterial culture were included retrospectively
in the efﬁcacy evaluation. Poor responders to the antimicrobial therapy were included in the evaluation for the emergence of resistant variants. Patients in whom the duration of treatment with
PIPC was ≤3 days and those who concomitantly received other antimicrobial agents were excluded from the analysis.

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Aug. 20172-2. Methods of evaluation of the efﬁcacy and the emergence of resistant variants
The efﬁcacy evaluation and evaluation for the emergence of resistant variants were based on
bacteriological evaluation. In the efﬁcacy evaluation, patients in whom the post-PIPC treatment
bacterial culture demonstrated disappearance of the isolate were regarded as responders, andthose in whom the post-PIPC culture showed persistence of the isolate were labeled as non-responders. In the evaluation for the emergence of resistant variants, the occurrence of an isolate
from a non-responder for which the MIC of PIPC increased from the initial susceptible range tothe intermediate or resistant range after the start of treatment was deﬁned as emergence of a resistant variant. The CLSI criteria were employed for the assessment of the antimicrobial susceptibility of the bacterial isolates8). The upper limit of detection of PIPC in the MIC assay at this hospital is 64 mg/L, so that it was impracticable to carry out the evaluation of the isolates for which theinitial MIC was already 64 mg/L; therefore, such isolates were excluded from the evaluation for
the emergence of resistant variants.
2-3. Calculation of %fT＞MIC
Changes in the serum PIPC concentrations over time in the subject patients were estimatedfrom results of the population pharmacokinetics (PPK) analysis7), and the %fT＞MIC was calculated for each bacterial isolate species from the changes of the serum concentrations over time
and the MIC for each isolate, using the programming software R, version.2.11.1. The changes inthe serum concentrations over time were calculated using the following formulae and also thebasic information of the subjects.
Conc＝{Dose/(CL·Tinf)}{exp(−kt*)−exp(−kt)}×fuB 0,( t≤Tinf )t* ＝ 
 t － Tinf , ( t >Tinf )k＝CL ⁄ Vd

fuB＝fraction of unbound drug in the blood

Using a protein-binding rate of PIPC of 21.2% (cited from Interview Form), the percent fraction of unbound drug in the blood was calculated. Meanwhile, data from the PPK analysis wereused for calculation of the clearance (CL) and distribution volume (Vd). Calculation of the CL requires the creatinine clearance (Ccr), which was derived from patientʼs basic information using
the Cockcroft-Gault equation16). For patients with a serum creatinine (Scr) of ＜0.6 mg/dL, inwhom the Ccr could be overestimated, the Scr was corrected to 0.6 mg/dL to calculate the Ccr. As
the lower detection limit of PIPC in the MIC assay is 8 mg/L at this hospital, the %fT＞MIC for
the organisms for which the MIC was ≤8 mg/L was calculated assuming an MIC＝8 mg/L. Similarly, for the organisms for which the MIC was ≥64 mg/L, the %fT＞MIC was calculated assuming an MIC＝64 mg/L, as the upper detection limit of PIPC in the MIC assay at our hospital isAug. 2017

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191（ 5 ）64 mg/L.
2-4. Statistical processing
Statistical analyses were performed using the Chi-squared test for categorical variables andthe Studentʼs t-test for continuous variables. Using logistic regression analysis, the “response rate
(good＝1, poor＝0)” was incorporated as a dependent variable and the %fT＞MIC as the independent variable in the efﬁcacy evaluation. In the evaluation for the emergence of resistant variants, the “percent emergence of resistant variants (resistant＝1, not resistant＝0)” was incorporated as a dependent variable and the %fT＞MIC as the independent variable. A p-value of ≤5%
was considered to denote statistical signiﬁcance. All statistical calculations were performed usinga software package (SPSS® version.20.0 for Windows®).
2-5. Ethical approval
This study was conducted with the approval of the Ethics Committee of Kitasato University
School of Medicine (Approval Number: The Ethics Committee of Kitasato University School of
Medicine and Hospital, B Ethics 09–80).

3. Results
3-1. Patients
Of the 190 patients treated with PIPC, the background characteristics of the 45 patients included in the efﬁcacy evaluation and 22 patients evaluated for the emergence of resistant variants
are summarized in Table 1. There were no signiﬁcant differences in the patient background characteristics between patients included in the efﬁcacy evaluation and patients included in the evaluation for the emergence of resistant variants.
3-2. Bacterial isolates
Details of the 56 strains of Gram-negative bacteria isolated from the patients included in the
efﬁcacy evaluation are shown in Figure 1. A breakdown of the 22 Gram-negative bacterial strains
included in the evaluation for the emergence of resistant variants is given in Figure 2. The bacterial isolates subjected to the analysis did not include any that produced extended-spectrum betalactamase (ESBL).
3-3. Relation of the efﬁcacy and emergence of resistant organisms to the %fT＞MIC
The present study results are schematically illustrated in Figure 3. Of the 56 Gram-negative
bacterial strains isolated from the patients included in the efﬁcacy evaluation, 32 strains proved tobe responsive to the treatment (mean %fT＞MIC, 38.3±14.6%). The remaining 24 strains were

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Table 1. Characteristics of patients included in this study

Fig. 1. Details of the 56 strains of Gram-negative bacteria isolated from the patients included in
the efﬁcacy evaluation

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193（ 7 ）Fig. 2. Details of 22 strains of Gram-negative bacteria isolated from the patients included in theevaluation for the emergence of resistant variants

Fig. 3. The present study results are schematically illustrated
From among inpatients treated with PIPC, those with bacterial isolates for which the MICs of PIPC were determined at thetime of bacterial culture were included retrospectively in the efﬁcacy evaluation. Poor responders to the antimicrobial therapy
were included in the evaluation for the emergence of resistant variants. In the evaluation for the emergence of resistant variants,
the occurrence of an isolate from a non-responder for which the MIC of PIPC increased from the initial susceptible range to theintermediate or resistant range after the start of treatment was deﬁned as emergence of a resistant variant. The CLSI criteria
were employed for the assessment of the antimicrobial susceptibility of the bacterial isolates.

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Fig. 4. Logistic regression analysis to determine the relationship between the efﬁcacy and%fT＞MIC

unresponsive to treatment (mean %fT＞MIC, 23.1±16.9%). Results of logistic regression analysis to determine the relationship between the efﬁcacy and %fT＞MIC are presented in Figure 4. A
model formula of therapeutic response rate＝1/[1＋exp(1.945−0.073*%fT＞MIC)] was estimated (P＜0.01), and the %fT＞MIC required for achieving a therapeutic response rate of 90%
was calculated as 56.7%. Of the 24 strains rated as unresponsive to the treatment in the efﬁcacy
evaluation, 22 were subjected to the evaluation for the emergence of resistant variants. Of these
22 Gram-negative strains, no resistant variants were found for 15 of the strains (%fT＞MIC, 38.2
±10.4%); on the other hand, resistant variants emerged for 7 of the strains (%fT＞MIC, 27.3±5.0%). Results of logistic regression analysis to determine the relationship between the emergence of resistant variants and the %fT＞MIC are presented in Figure 5. A model formula of therapeutic response rate＝1/[1＋exp(−5.855＋0.208*%fT＞MIC)] was estimated (P＜0.05), and the%fT＞MIC required for achieving a non-emergence rate of resistant variants of 90% was calculated as 38.7%. As a results of sub-analysis for each disease, although there is no signiﬁcant,there was a tendency like that sufﬁcient efﬁcacy rate of 90% can be obtained with %fT＞MIC＝47.2% for urinary tract infection (P＝0.051) and %T＞MIC＝82.6% for pneumonia (P＝0.176).

4. Discussion
Severe infections are associated with increased mortality rates, prolongation of hospitalization, and increase in medical expenses17,18). Gram-negative bacterial infections, in particular, areAug. 2017

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195（ 9 ）Fig. 5. Logistic regression analysis to determine the relationship between the emergence ofresistant variants and the %fT＞MIC

difﬁcult to treat and are associated with high mortality rates19). Furthermore, changes in the PK
proﬁles of drugs may also occur in patients with infections20). Increased exposure to drugs due tochanges in the PK of a drug increases the possibility of adverse drug reactions to the drug, whilst
decreased exposure to drugs increases the chances of failure of treatment or of emergence ofdrug-resistant organisms21). Therefore, selection of a dosing schedule appropriate for each patient
is of crucial importance. The trend of increasing resistant organisms is a major global publichealth concern, and apprehensions over the emergence of resistant organisms are especially relevant at medical facilities. Multidrug-resistant organisms have a bearing on the morbidity rate andduration of hospitalization, and may ultimately account, at least in part, for an increase in themortality rate. Therefore, effective methods are being sought to prevent transmission of resistantorganisms from infected patients to other patients, including by administration of appropriate antimicrobial agents at appropriate doses for appropriate periods22,23).
In the present study, logistic regression analysis revealed a signiﬁcant relationship of the%fT＞MIC to the efﬁcacy (bacteriological effect). The therapeutic response rate and the incidence of emergence of resistant variants were calculated for each level of %fT＞MIC, using thecomputed model formula (Table 2). Analysis using the model formula revealed that the therapeutic response rate was as high as 50% or more at %fT＞MIC＝30%, at which PIPC is known toexert a growth-inhibitory effect on Gram-negative bacteria, and that the therapeutic response rate
was ≥80% at %fT＞MIC＝50%, at which the drug is known to exert its maximal bactericidal effect. It follows that maintaining the %fT＞MIC at 30% would yield therapeutic response rates of196（ 10 ）THE JAPANESE JOURNAL OF ANTIBIOTICS

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Table 2. Relationship between the therapeutic response rate and the incidence of emergence ofresistant variants≥50%; while it was inferred from the results of analysis of the relationship between the emer-

gence of resistant variants and the %fT＞MIC, that the %fT＞MIC at which 50% of the infecting
organisms might acquire resistance in case of failure of antimicrobial drug therapy was 28.2%.
The present study represents a new research attempt to assess the clinical outcome by focusing on
the therapeutic response rate and the incidence of emergence of resistant variants with reference
to the %fT＞MIC, using clinical data. The results of our analysis showed that not only the therapeutic response rate, but also the incidence of emergence of resistant variants became worse at abacteriostatic level of the %fT＞MIC, suggesting the importance of selecting a dosing method
taking into account bactericidal levels of the %fT＞MIC. By designing the dosage aimed at attaining a %fT＞MIC of 60%, a therapeutic response rate of 90.5% and incidence of emergence ofresistant variants of 0.1% can be obtained, enabling sufﬁcient efﬁcacy and sufﬁcient preventionof emergence of resistant organisms.
With a view to interpreting the secured data by linking the dosing method to the clinical responses, probability of target attainment (therapeutic response rate and incidence of resistant variants) in various dosing programs was calculated for each level of renal function as well as foreach graded MIC level (8 mg/L, 16 mg/L, and 32 mg/L), with the drip infusion time set at 0.5
hour; the results are summarized in Tables 3-1, 3-2, and 3-3. The breakpoint for PIPC is set at
16 mg/L according to the CLSI and EUCAST criteria, whereby MIC＝16 mg/L is interpreted as
implying the organism is susceptible8,9). At MIC＝16 mg/L, it is considered that a therapeutic response rate of ≥90% can be attained by adopting a PIPC dose level of ≥12 g/day, even when the
Ccr＝100 mL/min (Table 3-2). At MIC＝32 mg/L, on the other hand, there is the possibility that a
sufﬁcient therapeutic response rate may not be obtained, depending on the severity of renal dys-

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197（ 11 ）Table 3-1. Probability of target attainment in various dosing programs for each level of renal
function (MIC＝8 mg/L)

Table 3-2. Probability of target attainment in various dosing programs for each level of renal
function (MIC＝16 mg/L)

function, even at a PIPC dose of 12 g/day (Table 3-3). These present ﬁndings provide evidence tosupport the deﬁnition of intermediate resistance by the CLSI. At MIC＝16 mg/L, therefore, treatment with PIPC is quite feasible and the breakpoint set in the CLSI and EUCAST criteria is considered to be valid.

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Table 3-3. Probability of target attainment in various dosing programs for each level of renal
function (MIC＝32 mg/L)

A limitation of this study was that cases with MIC ≤8 mg/L were considered as cases with
MIC＝8 mg/L for calculation of the %fT＞MIC, because the lower limit of detection of the MIC
assay for PIPC at our hospital is 8 mg/L. According to a surveillance reported by Tsuji et al., thepercentage of Pseudomonas aeruginosa isolates for which the MIC of PIPC is ＜8 mg/L is 44.4%,
suggesting the presence of clinical isolates of P. aeruginosa for which the MIC of PIPC is lessthan 8 mg/L24). Finally, there is the possibility that the MICs were overestimated in our present
analysis, resulting in underestimation of the %fT＞MIC. It would be reasonable to assume that
PIPC was effective against those isolates for which the MIC of the drug was ＜8 mg/L. Considering that the %fT＞MIC values may have been underestimated in this study, it is possible that the%fT＞MIC actually required to achieve a therapeutic response rate of 90% may be smaller thanthat estimated in this study. Further, the %fT＞MIC was calculated from the changes in the serum
drug concentrations over time, which were based on a renal function parameter (Ccr). However,it can be assumed that the changes in the serum drug concentrations over time and the therapeutic
response would vary with the drug distribution to the sites of infection and the severity of the infection. No assessments by the site/severity of the disorder were performed, nor were any assessments made taking into account the drug distribution into the infected tissues in the present analysis. Furthermore, the bacterial isolates that were evaluated for the emergence of resistant variants
were from the same patients and belonged to the same strains, however, no genetic assessment of
the strainsʼ identity was performed. This limitation was related to the retrospective nature of thestudy, and further prospective studies in a larger number of clinical cases are needed.

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199（ 13 ）A rise in the maximum dose of PIPC was approved in March 2015 in Japan, enabling clinical use of this drug at doses comparable to those used in Europe and the United States. It is considered deeply signiﬁcant for appropriate therapy in the future, that associations were demonstrated in the present analysis, carried out based on the PK/PD theory, between the response/
suppression of emergence of resistant variants and the dosage and administration schedule of antimicrobial drugs.
Conﬂict of interest
None declared.

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