A microbiological method for determining serum levels of broad spectrum
-lactam antibiotics in critically ill patients

Jimmy Fridlund, Hanna Woksepp, Thomas Schon

PII: S0167-7012(16)30192-0
DOI: doi: 10.1016/j.mimet.2016.07.020
Reference: MIMET 4966

To appear in: Journal of Microbiological Methods

Received date: 20 May 2016

Revised date: 22 July 2016
Accepted date: 23 July 2016

Please cite this article as: Fridlund, Jimmy, Woksepp, Hanna, Schon, Thomas,
A microbiological method for determining serum levels of broad spectrum -lactam
antibiotics in critically ill patients, Journal of Microbiological Methods (2016), doi:
10.1016/j.mimet.2016.07.020

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 ACCEPTED MANUSCRIPT

A microbiological method for determining serum levels of broad spectrum

-lactam antibiotics in critically ill patients

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Jimmy Fridlund1, Hanna Woksepp1,2, Thomas Schn1,2,3*

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1
Department of Clinical Microbiology, Kalmar County Hospital, Kalmar, Sweden
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Department of Medicine and Optometry, Linnaeus University, Kalmar, Sweden.
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Department of Medical Microbiology, Linkping University, Linkping, Sweden.

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REVISED VERSION
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*Corresponding author:
Thomas Schn
Dept. of Clinical Microbiology, Kalmar County Hospital
SE-391 85 Kalmar, Sweden
Tel: +4648081000, Email:tschon@hotmail.com

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Abstract

Background: Recent studies show that suboptimal blood levels of -lactam antibiotics are

present in intensive care unit (ICU) patients. A common reference method for assessing drug

concentrations is liquid chromatography coupled with mass-spectrometry (LC-MS) which is

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highly accurate but rarely available outside reference centres. Thus, our aim was to develop a

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microbiological method for monitoring -lactam antibiotic serum levels which could be used

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at any hospital with a microbiological laboratory.

Methods: The method was developed as a 96-well broth microdilution format to assess the

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concentrations of cefotaxime (CTX), meropenem (MER), and piperacillin (PIP). Patient

serum containing antibiotics were diluted in suspensions of bacteria with known minimal
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inhibitory concentrations (MICs). Serum antibiotic concentrations were calculated by dividing

the MIC with the dilution factor at which the serum inhibited growth of the bacterial
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suspension. Serum (n=88) from ICU patients at four hospitals in south-east Sweden were
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analysed and compared to LC-MS analysis.

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Results: The overall accuracy and precision for spiked samples and patient samples was
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within the pre-set target of 20.0% for all drugs. There was a significant correlation between
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the microbiological assay and LC-MS for the patient samples (CTX: r= 0.86, n=31; MER: r=

0.96, n=11; PIP: r= 0.88, n=39) and the agreement around the clinical cut-off for CTX (4.0

mg/l), MER (2.0 mg/l) and PIP (16.0 mg/l) was 90%, 100% and 87%, respectively.

Conclusion: The microbiological method has a performance for determination of serum

levels of meropenem, piperacillin and cefotaxime suitable for clinical use. It is an inexpensive

method applicable in any microbiology laboratory.

Keywords: -lactam antibiotics, cefotaxime, meropenem, piperacillin, microbiological

method, drug concentration.

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Introduction

The suggested target concentration for -lactam antibiotics in patients who are not critically ill or

immunosuppressed is at least 50% of the exposure time (T) for the free (f) fraction above the

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minimal inhibitory concentration (MIC) (50% f T>MIC) (Roberts et al., 2014). For patients admitted to

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the intensive care unit (ICU), a number of studies indicate that a target of 100% f T1-4x>MIC is required

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for maximum clinical efficacy and to limit development of resistance (Dulhunty, et al., 2013; Wong

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et al., 2014; Donadello et al., 2015). According to the DALI study (Roberts et al., 2014),

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concentrations of -lactams below 100% f T>MIC in critically ill patients were present in about 30%

of patients treated with meropenem (MER) and piperacillin (PIP).

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The main methods available for measuring antibiotic concentrations in patients are high pressure

liquid chromatography (HPLC) or liquid-chromatography coupled with tandem mass spectrometry

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(LC-MS/MS) (Carlier, et al., 2015). Alternative methods, such as immunoassays (Grange et al.,
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2014), and microbiological assays are available for some antibiotics, such as vancomycin (Rybak et
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al., 1991), teicoplanin (Erickson, et al., 1989) and ceftriaxone (Dafale et al., 2012). In general, LC-
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MS/MS based methods are used for therapeutic drug monitoring at reference centres (Mller and

Rentsch, 2010; Wong, et al., 2014). This method is highly accurate and able to assess multiple
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compounds in a single sample. It is however, not readily available to most hospitals due to high cost

and the maintenance requirements (Suthakaran and Adithan, 2006).

In Europe, susceptibility testing of bacteria by using a MIC determination method has been

harmonized by EUCAST (EUCAST, 2003). A modified version of this method could also be used to

assess antibiotic concentrations from human samples. Some examples are the studies by Steinmann

et al. (2010) and Candejas-Bueno (2012), where the concentration of the antifungal drugs

voriconazole and posaconazole were assessed by adding these drugs to wells in agar plates covered

with microbes. The concentrations were determined by the size of the clear area around the well. To

our knowledge, a microbiological method for assessing the concentrations of -lactam antibiotics for
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quantitative clinical assessment in patient serum samples has not previously been described and

validated. The purpose of a microbiological method would be to achieve a performance adequate for

clinical monitoring of antibiotic concentrations in patients where more accurate LC-MS methods are

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not available. Due to the increasing need for individualizing antibiotic treatment in critically ill

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patients based on drug concentrations, our aim was to develop a microbiological method for

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monitoring cefotaxime (CTX), meropenem and piperacillin serum levels which could be used at any

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hospital with a clinical microbiological laboratory.

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Material and Methods
Selection of bacterial strains
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The bacterial strains used in the microbiological assay were selected based on the MIC values

quality control ranges established by EUCAST (EUCAST, 2003). The main selection criteria for
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bacteria were the likelihood of incorporating the MIC when creating dilution series in order to most
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accurately identify concentrations around the clinical target MIC of each antibiotic (CTX: 4.0 mg/L;

MER: 2.0 mg/L, and PIP: 16.0 mg/L) (Roberts et al., 2014). Strains were also selected based on their
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speed of growth, stability and required growth conditions. Two bacterial species were used for CTX

and MER (Table 1); one with a high and one with a low MIC for these drugs in order to cover the
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target range for analysis in one run. For PIP, one bacterium was used, and the serum sample was

analysed undiluted and in a 1:2 dilution to cover a wider concentration range. The strains were stored

in aliquots at -70C in cryoinstant tubes (Scharlau; Barcelona, Spain). Before experiments, the

bacteria were grown on blood agar plates for 20 1h under aerobic conditions at 35-37C and

propagated for a maximum of five passages.

Preparation and storage of antibiotics and internal controls

Antibiotic stock solutions were prepared using PIP, MER, and CTX from Sigma Aldrich (St.

Louis, US-MO) (Purity by HPLC: CTX: 100.0%; MER: 99.8%; and PIP: 97.5%) diluted in sterile

distilled water to a final concentration of 5120 mg/l and stored at -70C for a maximum of 12

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months. The internal controls for CTX (4mg/L), MER (2mg/L) and PIP (16mg/L) were diluted in

Mueller-Hinton broth (MHb; DIFCO, Becton Dickinson, USA), aliquoted on a single occasion and

were kept in storage at -70C until used, with a maximum storage period of 12 months.

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Methodological outline for determining concentrations of cefotaxime, piperacillin and

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meropenem by the microbiological assay

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The microbiological assay was developed in a 96-well, broth-microdilution format. All solutions

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and patient samples used were equilibrated to room temperature prior to their use. The selected

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bacteria where MICs were predefined for the antibiotic to be tested (Table 1) in which the serum

samples were diluted, were included in each experiment (for experimental plate setup, please refer to
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supplementary method: Figure 1). In order to assess the serum antibiotic concentration, serum

samples were diluted using MHb containing the bacteria selected for the antibiotic to be tested
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(Table 1). After incubation, the concentration of each serum sample was calculated by dividing the
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MIC with the serum dilution factor inhibiting bacterial growth. The MIC used for detection was

determined in each experiment using an MIC control dilution series (MICControl) (supplementary
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method: Figure 1).

Determination of cefotaxime, meropenem and piperacillin using the microbiological assay

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Set-up for sample analysis

Each serum sample was analysed using two separate 96-well plates using two different bacteria to

cover for the high (SerumConc.S.aureus for CTX and SerumConc.P.aeruginosa for MER), and the low

concentration range (SerumConc.E.coli-CTX for CTX and SerumConc.E.coli-MER) depending on the

MICs. For PIP, the same bacterium was used for verifying both high and low concentrations

(SerumConc.E.coli-PIP-1:1; SerumConc.E. coli-PIP-1:2) in order to cover the analytical range (4-96 mg/l).

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Preparation of controls and serum samples

As controls, both the MIC determination of the bacteria used for the assay (MICControl) as well as

an internal control with a fixed concentration for each antibiotic diluted in MHb were included. To

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prepare the MICControl, the stock solution of the antibiotic to be determined (5120mg/l) was further

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diluted to the starting concentration indicated for the MICControl dilution in supplementary method:

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Tables 1-5. Then, the MICControl was dispensed in 1.5 ml tubes according to the dilution strategy in

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supplementary method: Tables 1-5. For the serum samples, one part was diluted 1:5 in MHb for CTX

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and MER and 1:2 for PIP for analysis of the higher concentration ranges. The undiluted part of the

serum sample used for the lower concentration ranges as well as the diluted serum sample and the
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undiluted internal control were then dispensed in 1.5 ml tubes (for specific volumes depending on

which antibiotic to be analysed, please refer to supplementary method: Tables 1-5).

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Preparation of bacterial suspensions

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Before analysis, the bacteria as described in Table 1 were grown on blood agar plates for 201h
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under aerobic conditions at 35-37C. Bacterial colonies were dissolved in 0.9% NaCl (Merck, NJ,
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U.S) to 0.5 McF by using a Densichek plus spectrometer (BioMrieux SA, France). The solution was

subsequently diluted 1:100 in MHb, to provide an approximate final concentration of 1*105 colony
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forming units (CFU) (EUCAST, 2003). The bacterial suspensions were then added to the MICControl

and the serum samples according to the dilution schemes in supplementary method: Tables 1-5

depending on the antibiotic to be analysed.

Outline for dispensing samples on the 96-well microtiter plate for analysis

Tubes with bacterial suspension and antibiotics including the MICControl (in duplicate) were mixed

vigorously with vortex agitation before 100 l was added to a 96-well microtiter plate (Sarstedt,

Nmbrecht, Germany) as outlined in supplementary method: Figure 1. The bacterial suspension

without antibiotics was used as positive control. MHb without bacteria was used as negative control.

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Plates were covered with adhesive film (Sigma Aldrich St. Louis, US-MO) and incubated for 201 h

at 35-37C before being analysed by visual inspection to determine the MICs. To pass the quality

control, we predefined that the internal control in each experiments should be within 20.0% of the

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target (2.8-5.2 mg/l for CTX, 1.4-2.6 mg/l for MER and 11.2-20.8 mg/l for PIP) similar to described

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by Candejas-Bueno et al. (2013). Additionally, the MICControl used for each assay had to be within

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one MIC dilution (EUCAST, 2003). If these conditions were not fulfilled, the analysis results were

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discarded and the sample was re-analysed.

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The concentration of the patient samples retrieved in each of the two 96-well plates were

calculated by dividing the plate specific MIC (mean of the two MICControl series), with the dilution
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factor of the first well lacking growth in the patient sample dilutions. The final concentration of the

serum sample was then determined from the concentrations in each of the two both 96-well plates in
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accordance with the flow charts presented in supplementary method: Figures 2-4.
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Serum concentrations of cefotaxime, meropenem and piperacillin in clinical samples clinical

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validation of the microbiological assay versus LC-MS

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In order to estimate the rate of patients achieving 100% f T>MIC, serum samples from ICU patients

participating in a previous, multi-centre prospective clinical study in south-east Sweden (Kalmar

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County Hospital; Linkping University Hospital; Vxj Central Hospital; and Jnkping County

Hospital Ryhov) were analysed (CTX n=31; MER n=18; PIP n=39). Samples were collected

immediately prior to administration of the next antibiotic dose. The study and the analysis of

antibiotic concentrations in patient serum were approved from the ethical committee at Linkping

University (Dnr 2014/236-31). The final protocol was used to assess the concentration in clinical

samples from ICU patients (CTX: n=31; MER: n=18; and PIP: n=39) stored at -70C for a maximum

of 12 months. The concentrations attained were compared to those attained previously, using LC-MS

in order to evaluate the accuracy and precision of unknown clinical samples. Analysis of PIP, MER

and CTX concentrations by LC-MS was performed at the Department of Clinical Pharmacology,

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Karolinska University Hospital, Stockholm, Sweden which is an accredited laboratory. Method

accuracy and precision for LC-MS are within +/-15 % for all drugs and the measurement range is

0.2-100 mg/L for PIP and 0.2-50 mg/L for MER and CTX.

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Statistics

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Statistical analysis of the correlation between the results of the microbiological assay and the LC-

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MS reference assay was done by using Spearman Rank Correlation test, in GraphPad Prism version 6

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for Windows, (GraphPad Software, San Diego California USA). In order to assess the accuracy and

precision of the microbiological method, RE% and the CV% was calculated. RE% was calculated
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using the sample deviation from the reference-concentration (i.e. LC-MS-concentrations for the

patient serum samples, and the known concentration in the case of spiked samples) divided by the
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reference-concentration.
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Results

Stability of MIC determinations for bacterial control isolates throughout the experiments

Throughout the method development and clinical sample analysis, the MICControls were recorded

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and compared to the MICs pre-defined by EUCAST for the ATCC strains used (Table 1). None of

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the antibiotics showed more than a 6% (CTX: n= 12/203; MER: n= 5/221; and PIP: n= 7/235)

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deviation beyond 1 two-fold MIC-dilution steps from the target MIC range (CTX: 2-8 mg/l; MER:

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1-4 mg/l; and PIP: 8-32 mg/l) (Table 1). In case the MIC was out of the target range, the analysis was

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declared invalid, and re-analysed.

Performance of the microbiological assay compared to LC-MS for ICU patient serum samples
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The overall inter-assay variation RE% and CV% were within the pre-set target of 20% for all

three antibiotics when using spiked serum samples (please refer to validation supplement for details).
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In clinical serum samples, the comparison between the microbiological assay and LC-MS showed
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that the RE% and CV% results for all three antibiotics were within the 20.0% cut-off limit (CTX:
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CV% = 9.5, RE%: = (-9.7); MER: CV% = 5.5, RE%: = 5.6; and PIP: CV% = 11.1, RE%: = (-19.4),
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supplementary validation: Table 1). Comparing the results of the microbiological assay to the

reference method revealed a strong correlation between the two methods for all three antibiotics
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(CTX: r= 0.86, p< 0.001, n = 31; MER: r= 0.96, p< 0.05, n = 18; PIP: r= 0.88, p< 0.01, n = 39)

(Figures 1A-C). The agreement between the reference method and the microbiological assay

regarding the concentration assessment around the clinical cut offs were 90 % (CTX = 28/31), 100 %

(MER = 18/18) and 87 % (PIP = 34/39) for the three drugs respectively.

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Discussion

We developed a microbiological method for determining serum concentrations of cefotaxime,

meropenem, and piperacillin which could be performed in any microbiological laboratory at a low

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cost. The method showed adequate performance within 20.0% for CV% and RE%, both when

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considering inter- and intra-assay variation. Thus, the accuracy and precision of the analysis is

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sufficient for clinical use in recommending dose adjustments if serum drug levels are below the cut-

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off.

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We found few, if any published microbiological methods for determining serum levels of

cefotaxime or piperacillin for clinical purposes although it was used by one centre in a recently
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published multi-centre survey on the use of therapeutic drug monitoring of -lactam antibiotics in

ICU patients (Wong, et al., 2014). For meropenem, a microbiological assay was described (Mendez
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et al. 2005) although not directly adopted for clinical use.

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Compared to the reference microdilution broth MIC method described by the EUCAST in which
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a 1:2 dilution is used, the microbiological method applies a narrower MIC-dilution, both for the
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bacterial isolate for diluting the serum sample as well as for the internal control and serum samples.

Although, the 1:2 dilution is standard for MIC testing in clinical practise, there was a need to create a
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more fine-tuned MIC dilution in order to accurately determine serum drug concentrations by using

the known MICs. As the dilution strategy was central in the method development we choose for

simplicity to dilute the serum samples as well as the bacteria. This strategy was regarded as

acceptable as the difference in the bacterial volume did not affect the final MIC-determination as

shown in the validation supplement.

Even if the microbiological assay has a performance which is inferior to LC-MS for determining

antibiotic concentrations, our results show that it is sufficient for the demands in clinical routine. In

the microbiological assay, we noted that there were systematic trends where CTX and PIP tends to

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be underestimated whereas MER is more likely to be overestimated (supplementary validation:

Tables 2-3). LC-MS is a more accurate and precise method which should be used where available

and it is also faster than the microbiological method as it can be analysed within the same day. In the

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microbiological method one drug concentration takes about 1h hands-on time but requires overnight

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incubation. However, the microbiological assay could be of importance outside reference centres

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where LC-MS is not available and sample transport may delay results on critically ill patients for at

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least 3-4 days.

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There are several reasons why the microbiological method is not as accurate as LC-MS. Small

differences during the preparation of the dilution series providing the MICControl for each plate might
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affect the results. Such potential variations might be caused by bacterial clumping and variations in

the bacterial inoculum. This risk is especially likely for S. aureus, as is noted by Cotar et al. (2010),
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as, due to specialized surface proteins, it is highly prone to clumping. The ability of P. aeruginosa to
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form adhesive biofilms may also contribute to higher-than-intended numbers of cells to the MHb-

bacteria mix. Another possibility is that, interference with bacterial growth from compounds in the
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patient sample, including parts of the immune system present in the sera such as antibodies and

components of the complement system may affect the results. In addition, despite the high
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correlation between the two methods (Figures 1A-C), there are occasional differences between the

microbiological assay and the LC-MS method. One explanation for this might be that the samples

had been analysed previously (3-9 months before) by LC-MS and the lower concentrations in the

microbiological assay could be explained by antibiotic degradation. Moreover, the readout in the

microbiological method is based on visual evaluation of the MIC in contrast to LC-MS which is

more objective.

Our method has several limitations. The major one is the inability to assess the drug concentration

of patients being treated using more than one antibiotic. Here, additional treatment with drugs which

affect the susceptibility of the bacteria used in the assay such as fluoroquinolones or aminoglycosides

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may overestimate drug concentrations. Thus, we recommend that the patients should be on single

therapy for more than 24 hours before the microbiological assay is used. This particular limitation

may require some continued work on this method, as multiple antibiotics is recommended as a

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treatment for severe pneumonia and sepsis (Wong, et al., 2014). In the study by Woksepp et al.

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(Submitted), approximately 30% of ICU patients in South-East Sweden were treated with more than

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one antibiotic.

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Nevertheless, the microbiological method could be an important contribution for hospitals without

access to a LC-MS method for determining broad spectrum -lactam drug concentrations in critically

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ill patients. However, the method should be prospectively validated against a reference method such
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as LC-MS for the purpose of therapeutic drug monitoring.

In conclusion, a microbiological method for determination of cefotaxime, piperacillin and

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meropenem has been developed for clinical use.

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Acknowledgements
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This study was supported by grants from the research council of Southeast Sweden (FORSS) and
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Marianne and Marcus Wallenberg foundation.

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Figures and Tables

Table 1: Bacterial ATCC strains chosen for the microbiological assay, together with the total agreement between the EUCAST MIC range and the recorded

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concentrations attained throughout the study. Total tests performed (n), along with the number of occasions where the observed MICControl were within the

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acceptable EUCAST MIC range.

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EUCAST EUCAST Agreement

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Bacterial > +1 MIC Within 1 MIC <(-1) MIC with
Antibiotic ATCC target MIC MIC range n
strain step step step

N
(mg/l) (mg/l) EUCAST

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S. aureus 29213 2.0 14
CTX 203 5 191 7 94.1%
E. coli 25922 0.125 0.064 0.25

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P. aeruginosa 27853 0.5 0.25 1

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MER 221 3 216 2 97.7%
E. coli 25922 0.016 0.008 0.032

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PIP E. coli 25922 2.0 14 235 7 228 0 97.0%
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M ic r o b io lo g ic a l A s s a y

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5

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L C - M S /M S
Figure 1A. Correlation plot of samples from ICU patients treated with cefotaxime analysed by LC-MS
and the microbiological assay respectively (p<0.0001. r= 0.86. n = 31). Squares = Samples analysed by
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diluting bacteria in normal human serum. Circles = Samples analysed by diluting bacteria in Mueller-
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Hinton broth.
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20
M ic r o b io lo g ic a l A s s a y

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20 30
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L C - M S /M S

Figure 1B. Correlation plot of samples from ICU patients treated with meropenem analysed by LC-
MS and the microbiological assay respectively (p< 0.0001. r= 0.96 n = 18). Squares = Samples
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analysed by diluting bacteria in normal human serum. Circles = Samples analysed by diluting bacteria
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in Mueller-Hinton broth.
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ACCEPTED MANUSCRIPT

M ic r o b io lo g ic a l A s s a y 200

150

T
IP
100

R
SC
50

0 NU
MA
0 50 100 150 200
L C - M S /M S
D

Figure 1C. Correlation plot of samples from ICU patients treated with piperacillin analysed by LC-
MS and the microbiological assay respectively (p< 0.0001. r= 0.88. n = 39). Squares = Samples
TE

analysed by diluting bacteria in normal human serum. Circles = Samples analysed by diluting bacteria
in Mueller-Hinton broth.
P
CE
AC

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 ACCEPTED MANUSCRIPT

Highlights

A microbiological method for determination of serum drug concentrations of piperacillin,

meropenem and cefotaxim was developed
In a comparison to LC-MS methodology as the reference, the precision and accuracy was
within 20%

T
R IP
SC
NU
MA
D
P TE
CE
AC

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