A Phase III Randomised Controlled Trial of Continuous Beta-lactam Infusion Compared With Intermittent Beta-lactam Dosing in Critically Ill Patients
Phase: NA
Conditions: Sepsis
Sponsor: The George Institute

The Beta-Lactam InfusioN Group 
BLING III Study PK-PD Substudy Protocol  
A phase III randomised controlled trial of continuous beta-lactam 
infusion compared with intermittent beta-lactam dosing in 
critically ill patients 
Protocol Number: TGI-CCT254643
Version Number: 6.0 
Date:  8 November 2022
ClinicalTrials.gov Register: NCT03213990

BLING III Study: PK-PD Sub-study Protocol   Version 6.0  8th November 2022 
 
 
2 
Table of Contents 
1. 
PROTOCOL SYNOPSIS 
3 
2. 
ADMINISTRATION INFORMATION 
5 
2.1. 
SUB-STUDY STEERING COMMITTEE 
5 
2.2. 
COORDINATING CENTRE AND CENTRAL BIOANALYSIS LABORATORY 
5 
2.3. 
FUNDING 
6 
2.4. 
ROLE OF FUNDING BODIES 
6 
3. 
BACKGROUND AND RATIONALE 
6 
4. 
STUDY DESIGN 
7 
4.1. 
AIM 
7 
4.2. 
MAIN OBJECTIVES 
7 
4.3. 
HYPOTHESES 
8 
4.4 STUDY DESIGN 
8 
4.5 PATIENT INCLUSION CRITERIA 
9 
5. 
STUDY PROCEDURE 
9 
5.1. 
PHARMACOKINETIC COMPONENT 
9 
5.2. 
PHARMACODYNAMIC COMPONENT  
10 
6. 
WITHDRAWAL FROM THE PK-PD SUB-STUDY 
12 
7. 
SAFETY CONSIDERATIONS 
12 
8. 
DATA ANALYSIS METHODS 
13 
8.1. 
SAMPLE SIZE 
13 
8.2. 
STATISTICAL ANALYSIS PLAN 
13 
9. 
DATA COLLECTION 
13 
10. 
QUALITY CONTROL AND QUALITY ASSURANCE 
13 
11. 
ETHICAL CONSIDERATIONS 
13 
12. 
PUBLICATION POLICY 
13 
13. 
PROPOSED PROJECT TIMELINE 
14 
14. 
REFERENCES 
15 
15. 
APPENDIX 1 
16 
 
 
 

BLING III Study: PK-PD Sub-study Protocol   Version 6.0  8th November 2022 
 
 
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1. Protocol synopsis 
Title  
Continuous vs intermittent infusion of β-lactam antibacterial drugs: 
impact on resistance and outcomes in sepsis 
Short title 
BLING III PK-PD sub-study 
Design 
This is a prospective, observational sub-study of the BLING III study. 
Main 
Objectives 
1. To compare target attainment in culture positive patients by 
randomisation status according to the following definitions: 
a) ‘Equivalent PK-PD target’ for beta-lactam antibiotic exposure, 
defined as 100%ƒT>MIC for both treatment arms 
b) ‘Variable PK-PD target’ for beta-lactam antibiotic exposure, 
defined as ƒCss≥4xMIC for the continuous infusion arm and 
100%ƒT>MIC for the intermittent infusion arm 
c) ‘Alternative variable PK-PD target’ for beta-lactam antibiotic 
exposure, defined as ƒCss≥4xMIC for the continuous infusion arm and 
50%ƒT>MIC for the intermittent infusion arm. 
2. To explore the association in culture-positive patients between beta-
lactam antibiotic exposure and the following outcomes by 
randomisation status: 
a) All-cause patient mortality at Day 90 post randomisation (primary 
outcome in the BLING III RCT) 
b) Patient clinical cure at Day 14 post randomisation (secondary 
outcome in the BLING III RCT) 
c) New acquisition, colonisation or infection with a multi-resistant 
organism or Clostridium difficile associated diarrhoea up to 14 
days post randomisation (secondary outcome in the BLING III RCT) 
 
Study 
procedures 
Pharmacokinetic component  
Three blood samples collected to determine unbound plasma antibiotic 
concentrations (termed ‘antibiotic exposure’). Samples collected 
between days 2 and 5 of beta-lactam antibiotic therapy. Pharmacokinetic 
analyses will be undertaken to describe beta-lactam antibiotic exposure 
in terms of: 
- 
unbound beta-lactam antibiotic concentration above the MIC for 
the entire dosing interval (i.e. ƒT>MIC) 
- 
24-hour area under the concentration-time curve of unbound beta-
lactam antibiotic to MIC ratio (i.e. ƒAUC0-24/MIC) 
Pharmacodynamic component 
For culture positive patients: Causative organism MIC will be 
preferentially determined via the broth microdilution and/or epsilometer 
test (Etest®) methods. Whole-genome sequencing will be performed on 
isolates of causative organisms to determine sequence types and 
characterise beta-lactamase genes (if applicable). 

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For culture negative patients: see Section 5.2 for further detail. 
Sample size 
300 culture-positive patients. Due to study design, culture negative 
patients will also be included in additional analyses. 
Patient 
inclusion 
criteria 
Patients enrolled into the BLING III study from sites participating in the 
PK-PD sub-study and for whom there is written consent to collect and use 
blood samples can be enrolled in the substudy if the following inclusion 
criteria are met: 
• Patient has either an arterial line or central venous catheter from 
which to sample blood from 
• 
Patient is able to be sampled between days 2 and 5 post 
commencement of beta-lactam antibiotic therapy. 
Site 
requirements 
Sites participating in the BLING III study meeting the following criteria will 
be invited to participate in the sub-study: 
1) Capacity to collect and process three timed blood samples 
2) Capability of microbiology laboratory to collect and transfer clinical 
culture isolate samples 
3) Provision to store aliquoted plasma samples and cultured isolates 
at -70oC to -80oC. 
 
 
 

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2. Administration information 
The Beta-Lactam Infusion Group (BLING) III pharmacokinetic-pharmacodynamic (PK-PD) sub-
study forms part of the BLING III randomised controlled trial (RCT). The BLING III Management 
Committee has overall responsibility for conduct of the BLING III trial and the PK-PD sub-study. 
A sub-study steering committee will provide expert advice on sub-study management. 
The BLING III PK-PD sub-study has received endorsement from the Australian and New 
Zealand Intensive Care Society (ANZICS) Clinical Trials Group, the Australasian Society for 
Infectious Diseases Clinical Research Network, the European Society of Clinical Microbiology 
and Infectious Diseases and the European Society for Intensive Care Medicine. 
2.1. Sub-study Steering Committee 
The BLING III PK-PD Sub-study Steering Committee will comprise the following Investigators 
with additional representatives invited as required (Chair followed by alphabetical order): 
• Professor Jason Roberts (CHAIR), Pharmacist Consultant, Royal Brisbane and Women’s 
Hospital, and Professor of Medicine, Faculty of Medicine, UQ Centre of Clinical Research, 
The University of Queensland, QLD, Australia  
• Dr Mohd Hafiz Abdul Aziz, Research Fellow, Faculty of Medicine, UQ Centre of Clinical 
Research, The University of Queensland, QLD, Australia 
• A/Prof Mark Chatfield, Senior Biostatistican, Faculty of Medicine, UQ Centre of Clinical 
Research, The University of Queensland, QLD, Australia 
• Dr Menino Osbert Cotta, Research Fellow, Faculty of Medicine, UQ Centre of Clinical 
Research, The University of Queensland, QLD, Australia 
• Professor Joshua Davis, Infectious Diseases Physician, John Hunter Hospital, NSW, 
Australia  
• Professor Jan De Waele, Surgical Intensivist, Department of Critical Care Medicine, Ghent 
University Hospital, Belgium 
• Dr Joel Dulhunty, Research Fellow, Department of Intensive Care Medicine, Royal 
Brisbane and Women’s Hospital, QLD, Australia 
• Dr Andrew Henderson, Infectious Diseases Physician & Clinical Microbiologist, Princess 
Alexandra Hospital, QLD, Australia 
• Professor Jeffrey Lipman, Director, Department of Intensive Care Medicine, Royal 
Brisbane and Women’s Hospital, QLD, Australia 
• Professor David Paterson, Infectious Diseases Physician, Royal Brisbane and Women’s 
Hospital, QLD, Australia 
• Ms Dorrilyn Rajbhandari (BLING III study PROJECT MANAGER), Critical Care & Trauma 
Division, George Institute for Global Health, NSW, Australia 
• Dr Steven Wallis, Laboratory Manager and Senior Scientist, Faculty of Medicine, UQ 
Centre of Clinical Research, The University of Queensland, QLD, Australia 
 
2.2. Coordinating Centre and Central Bioanalysis Laboratory 
Antimicrobial Optimisation Group 
Faculty of Medicine 
Level 8, UQ Centre for Clinical Research 
The University of Queensland 
Royal Brisbane and Women's Hospital, Herston QLD 4029 

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2.3. Funding 
The BLING III PK-PD sub-study has received part funding from the Royal Brisbane and Women’s 
Hospital Foundation. Etests® for minimum inhibitory concentration (MIC) are donated by 
bioMérieux (Etoile, France). The sub-study has been awarded a  United States Food and Drug 
Administration (FDA) Grant/Contract (Contract No. 75F40121C00126) commencing 
November 2021. Australian National Health and Medical Research Council (NHMRC) Centre 
of Research Excellence RESPOND (APP2007007) and a NHMRC Investigator Grant 
(APP2009736) to Professor Jason Roberts provide further funding to support the study. 
 
2.4. Role of funding bodies 
The BLING III PK-PD sub-study will be designed and conducted, and the results analysed, 
presented and published by the investigators independent of any funding agencies. 
3. Background and rationale 
Bacterial killing for beta-lactam antibiotics is related to the duration of time that bacteria are 
exposed to unbound antibiotic concentrations, or more accurately termed antibiotic 
exposure, that exceed the minimum inhibitory concentration (MIC) of the pathogen.1 Both in 
vitro and in vivo mathematical PK-PD models have provided a means to accurately describe 
beta-lactam antibiotic exposure targets that are associated with maximum effect.2 For the 
most part, these described PK-PD exposure targets have been supported by results reported 
in clinical studies.3 However, there is still some discordance in the literature on what 
constitutes optimal beta-lactam antibiotic plasma concentrations from the point of view of 
clinical cure and microbiological eradication.  
In some clinical studies, targets for beta-lactam antibiotic exposure associated with 
microbiogical eradication have been shown to correlate with unbound plasma concentrations 
maintained four to six times above the MIC throughout the entire dosing interval (i.e. 100% 
fT>4-6xMIC).4-6 Other pre-clinical data have shown maximal bactericidal effect occurs for only a 
proportion of, rather than the entire dosing interval. Based on this, PK-PD exposure targets 
for unbound concentrations remaining four times above the MIC (4xMIC) for 50%, 70% and 
40% for the penicillins, cephalosporins and carbapenems, respectively, have been stipulated.7 
Hence, a suggested target for beta-lactam antibiotic exposure of unbound plasma 
concentrations above the MIC for the entire dosing interval (i.e. 100% fT>1xMIC) has been 
advocated to ensure that 40-70% fT>4xMIC is achieved.8  
However, whether 100% fT>1xMIC ensures 40-70% fT>4xMIC has recently been challenged, with a 
recent study using existing patient data to simulate first doses suggesting that only a small 
number of patients will achieve 40-70% fT>4xMIC (for beta-lactam antibiotics such as 
piperacillin, ceftazidime and cefepime) with dosing to a therapeutic PK-PD target of 100% 
fT>1xMIC.9 These investigators strongly suggest an alternative exposure target of unbound 
plasma concentrations maintained four times above the MIC throughout the entire dosing 
interval (i.e. 100% fT>4xMIC) for intermittent infusion, and a steady-state concentration of the 
unbound antibiotic is at least four times greater than the MIC (i.e. ƒCss≥4xMIC) for continuous 
infusion. Based on existing data, they propose that this target will ensure maximal bacterial 
killing, prevent bacterial regrowth and ensure positive clinical outcome.  

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The 2014 DALI (Defining Antibiotic Levels in Intensive care unit patients) study showed that 
clinical outcomes were better when 100% fT>1xMIC was achieved as compared to lower 
concentrations.10 However, as this and other studies have not investigated outcomes 
associated with alternative PK-PD targets, such as 100% fT>4xMIC, it is currently unknown if 
higher PK-PD targets lead to incrementally better patient outcomes in the critically ill 
population. 
The large-scale recruitment during the BLING III study provides a unique opportunity for the 
collection of the largest and most comprehensive dataset of beta-lactam antibiotic PK-PD 
data. Serial blood samples and, if available, microbiological cultures will be collected. With this 
rich PK-PD dataset available, the BLING III PK-PD sub-study will be the best and perhaps, the 
only opportunity to describe the antibiotic exposure-clinical response relationship in the 
critically ill population. These data will define what method of administration (i.e., continuous 
infusion or intermittent infusion) and also, exposure (or concentration) of the beta-lactam 
antibiotic provide maximal therapeutic effect (patient survival) and also minimises the 
development of resistance in critically ill patients with sepsis. Additionally, by being conducted 
within the BLING III study, which is a pragmatic global trial, it will cost-effectively maximise 
generalisability across a range of infections, antibiotic resistance profiles and geographic 
regions/countries with different health care systems. 
 
4. Study design 
4.1. Aim 
The primary aim of the BLING III PK-PD sub-study is to determine the relationship between 
beta-lactam antibiotic exposure and patient-related outcomes observed in a subset of 
patients enrolled into the BLING III RCT. 
 
4.2. Main Objectives 
1. To compare target attainment in culture-positive patients by randomisation status 
according to the following definitions: 
a) ‘Equivalent PK-PD target’ for beta-lactam antibiotic exposure, defined as 
100%ƒT>1xMIC1 for both treatment arms2 
b) ‘Variable PK-PD target’ for beta-lactam antibiotic exposure, defined as ƒCss≥4xMIC3 
for the continuous infusion arm and 100%ƒT>1xMIC1 for the intermittent infusion 
arm2 
c) ‘Alternative variable PK-PD target’ for beta-lactam antibiotic exposure, defined as 
ƒCss≥4xMIC3 for the continuous infusion arm and 50%ƒT>1xMIC4 for the intermittent 
infusion arm5 
2. To explore the association in culture-positive patients between beta-lactam 
antibiotic exposure and the following outcomes by randomisation status: 
a) All-cause patient mortality at Day 90 post randomisation (primary outcome in the 
BLING III RCT) 
b) Patient clinical cure at Day 14 post randomisation6 (secondary outcome in the 
BLING III RCT) 

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c) New acquisition, colonisation or infection with a multi-resistant organism or 
Clostridium difficile associated diarrhoea up to 14 days post randomisation 
(secondary outcome in the RCT) [expected outcome prevalence << 15%]. 
APACHE = Acute Physiology and Chronic Health Evaluation. BLING = Beta-lactam Infusion Group. MIC = 
minimum inhibitory concentration. PK-PD = pharmacokinetic-pharmacodynamic. RCT = randomised controlled 
trial.  
1 - 100%ƒT>1xMIC = unbound beta-lactam antibiotic concentration above the MIC for the entire dosing interval. 
2 - For patients randomised to the continuous infusion arm, the mean value of the 3 steady-state 
concentrations will be used; for patients randomised to the intermittent infusion arm, the measured ‘trough’ 
concentration will be used. 
3 - ƒCss≥4xMIC = unbound beta-lactam antibiotic steady-state concentration four or more times above the MIC. 
4 - 50%ƒT>1xMIC= unbound beta-lactam antibiotic concentration above the MIC half-way (i.e. ‘mid’) through the 
dosing interval. 
5 - For patients randomised to the continuous infusion arm, the mean value of the 3 steady-state 
concentrations will be used; for patients randomised to the intermittent infusion arm, the measured ‘mid’ 
concentration will be used. 
6 - Clinical cure is defined as the completion of the beta-lactam antibiotic treatment course (on or before Day 
14) without recommencement of antibiotic therapy within 48 hours of cessation for the same infectious episode. 
For the purposes of evaluating clinical cure, change of antibiotic therapy (i.e. either escalation or de-escalation) 
for the same indication for which the beta-lactam antibiotic was commenced is considered part of the antibiotic 
treatment course 
 
4.3. Hypotheses 
For Objective 1: It is hypothesised that 1) attainment of the ‘Equivalent PK-PD target’ will be 
higher in patients randomised to the continuous infusion arm, 2) attainment for ‘Variable 
PK-PD target’ will be equivalent between the two treatment arms, and 3) attainment for 
‘Alternative Variable PK-PD target’ will be higher in patients randomised to the intermittent 
infusion arm. 
 
For Objective 2: It is hypothesised that the beta-lactam antibiotic exposure vs outcome 
curves will be similar for both continuous and intermittent infusion randomisation groups. 
Increasing beta-lactam antibiotic exposure will be associated with improved patient 
outcomes (clinical cure up to Day 14 and survival up to Day 90) up to a point, beyond which 
there may be a worsening in patient outcomes due to a potential association between very 
high beta-lactam exposure and increased sickness severity. The effect of confounding will be 
minimised by inclusion of relevant factors in regression models. It is hypothesised that 
increasing beta-lactam antibiotic exposure will also be associated with a decrease in the 
incidence of new acquisition, colonisation or infection with a multi-resistant organism or 
Clostridium difficile associated diarrhoea up to Day 14 
 
4.4 Study Design 
This is a prospective, observational PK-PD sub-study of the BLING III study.  
 
BLING III is a prospective, multi-centre, open, phase III, randomised controlled trial 
comparing continuous infusion of a beta-lactam antibiotic (piperacillin-tazobactam or 
meropenem) with intermittent infusion of the beta-lactam antibiotic over 30 minutes in 
critically ill patients with infection. 
 

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4.5 Patient inclusion criteria 
All patients enrolled into BLING III at sites participating in the BLING III PK-PD sub-study are 
eligible for inclusion into this substudy if the following inclusion criteria are met: 
• Patient has either an arterial line or central venous catheter from which to sample 
blood from 
• Patient is able to be sampled between days 2 and 5 post commencement of beta-
lactam antibiotic therapy. 
 
4.6 Participation requirements for sites 
Sites participating in the BLING III study meeting the following eligibility criteria will be invited 
to participate in the PK-PD sub-study: 
1) Capacity to collect and process three timed blood samples 
2) Capability of microbiology laboratory to collect and transfer clinical culture isolate 
samples 
3) Provision to store aliquoted plasma samples and culture isolates at -70oC to -80oC. 
5. Study procedure 
5.1. Pharmacokinetic component 
Sample collection 
Patients successfully enrolled into the sub-study will have three blood samples collected to 
determine unbound plasma antibiotic concentrations. Sampling will occur between days 2 and 
5 of beta-lactam antibiotic therapy. 
Blood collection tubes with heparin are required to be used for all blood sample collections. 
Once collected, the blood samples need to be placed in an ice-bath until centrifugation can be 
performed. Centrifugation needs to occur within 6 hours of blood sample collection. Plasma 
from centrifuged samples will be pipetted into a clean polypropylene cryovial, labelled with 
sub-study identifiers and stored at -70°C to -80°C. 
Patients randomised to the continuous infusion arm of the BLING III study: 
Blood samples will be collected 4 hours apart during the sampling window period between 
days 2 and 5 of antibiotic therapy (Figure 1). The continuous infusion dose must be running at 
a constant rate during the sampling period. 
Sample 1:  Time 0 (e.g. 0800 hrs) 
Sample 2:  Time 4 hrs (e.g. 1200hrs) 
Sample 3:  Time 8 hrs (e.g. 1600hrs) 
 
Patients randomised to the intermittent infusion arm of the BLING III study: 
Blood sampling will occur during one dosing interval (Figure 1). 
Sample 1:  30 minutes after the start of the intermittent infusion dose [‘0.5hr’] 
Sample 2:  Half-way through the dosing interval (i.e. if dosing interval is 8 hours, then 
sample will be taken at 4 hours) [‘Mid’] 

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Sample 3:  Prior to the next scheduled intermittent infusion dose (must be within 30 
minutes) [‘Trough’] 
 
 
 
Figure 1. Sampling times between days 2 and 5 of antibiotic therapy for continuous infusion 
and an intermittent infusion administered at a 8-hour dosing interval 
 
Sample analysis 
Beta-lactam antibiotic assays of blood samples will be conducted by the Central Bioanalysis 
Laboratory at UQ Centre of Clinical Research (UQCCR) at The University of Queensland to 
determine unbound antibiotic concentrations. Only samples where written consent has been 
obtained will be transferred for analysis. Any part of a sample remaining after analysis will be 
destroyed. Samples where no written consent is obtained will be destroyed at study sites. 
 
5.2. Pharmacodynamic component 
Microbiological cultures 
As part of routine clinical care at each participating site, biological samples will be sent to 
the clinical laboratory for microbiological culture. Viruses, fungi, parasites, rickettsiae and 
some fastidious bacteria (e.g., Legionella species, Brucella species and Leptospira species) 
are not considered relevant causative organisms in this sub-study. 
 
 

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“A causative organism” is defined as “a bacterium that is able to be grown by the participating 
site’s clinical laboratory” and must meet one of the following criteria: 
• 
Sterile site isolates (e.g., blood, bone, cerebrospinal fluid, deep fluid or pus, synovial fluid, 
and deep tissue): an organism which the treating team identifies as responsible for the 
current episode of sepsis. These should not include organisms generally considered to be 
contaminants, unless the organism is grown two or more times within a 72-hour period 
with identical antibiograms and the treating team identifies it as responsible for the 
current episode of sepsis 
• 
Sputum or endotracheal aspirates: a pure or predominant organism which is a known 
pathogen in a purulent sample with moderate or heavy growth, and is identified by the 
treating team as responsible for the current episode of sepsis 
• 
Urine samples: pure or predominant growth of at least 105 colony forming units per mL, 
and at least 10 white blood cells per high power field, and identified by the treating team 
as responsible for the current episode of sepsis 
• 
Other samples: an organism grown from another non-sterile site may be included if the 
treating team identifies it as responsible for the current episode of sepsis. 
 
For new acquisition, colonisation or infection with an MRO or Clostridium difficile associated 
diarrhoea up to 14 days post randomisation: a patient will be considered to have a relevant 
MRO based on defined clinical criteria and an identified organism (only bacterium) if cultured 
from samples taken between Day 1 and Day 14 post-randomisation (inclusive). 
 
MIC determination 
Isolates from causative organisms will be sent to UQCCR for formal susceptibility testing (by a 
Microbiological Research Scientist). Integrity of the isolate will be maintained by transfer of a 
10 µL loop of culture into a labelled vial containing glycerol (one organism per vial) which will 
be stored at –70oC to –80°C before transfer to UQCCR. A commercial courier company 
specialising in transport of clinical samples on dry ice will collect the microbiological isolates 
from each site and deliver to Central Bioanalysis Laboratory at UQCCR (Brisbane, Australia) for 
microbiological analysis. Once received, the MIC of the organism will be determined by broth 
microdilution (BMD); as the gold standard and to provide maximum accuracy for antibacterial 
susceptibility testing and E-test® (to provide generalisability for an alternative susceptibility 
testing method which is more commonly used in clinical laboratories). Our approach will 
provide maximum accuracy for defining the β-lactam antibacterial concentration to MIC ratio 
from which achievement of the PK-PD exposure can be preferentially determined, both for 
BMD and E-test® methods of antibacterial susceptibility testing. When more than one 
causative organism has been identified, MICs will be determined for each causative organism. 
The highest MIC will be imputed into beta-lactam antibiotic exposure calculations. Where an 
additional antibiotic is prescribed for a pathogen which is non-susceptible to piperacillin-
tazobactam or meropenem, the highest MIC of the other pathogen/s will be used (e.g. 
vancomycin co-prescribed with meropenem for a patient with cultures of both methicillin-
resistant S. aureus and P. aeruginosa, the P. aeruginosa MIC would be used for defining PK-
PD exposure for meropenem). 
 
For culture negative patients: median MIC values for piperacillin-tazobactam and 
meropenem based on culture-positive patient data will be used (see ‘Additional analyses’ in 

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12 
Appendix 1 for further detail). Additionally, analyses will be undertaken whereby MIC values 
in culture-negative patients will be based on MIC clinical breakpoints based on Pseudomonas 
aeruginosa susceptibility (i.e. 16 mg/L for piperacillin-tazobactam and 2 mg/L for 
meropenem). 
 
Identification of beta-lactam antibiotic resistance genes 
Whole-genome sequencing will be performed on isolates of causative organisms to determine 
sequence types and characterise beta-lactamase genes. Genomic DNA will be extracted using 
UltraClean DNA isolation kits (MoBio, Australia) and QIAmp DNA mini kits (Qiagen, Australia) 
and quantified by spectrophotometry (NanoDrop, ThermoFisher) and fluorometry (Qubit, 
ThermoFisher). Paired-end DNA libraries will be prepared using Nextera kits (Illumina, 
Australia). Whole-genome sequencing will be performed at the Central Bioanalysis Laboratory 
at UQCCR (Brisbane, Australia) by a Microbiological Research Scientist, using Illumina HiSeq 
(100 bp paired end) and MiSeq (300 bp paired end), as well as Illumina NextSeq (150 bp paired 
end) at the Forensic and Scientific Services Laboratory. Strains are also checked for 
contamination using Kraken (v1.1). Antibacterial resistance genes will be detected using 
assemblies and Abricate (v0.8) against the ResFinder and ARG-ANNOT databases. Resistance 
genes are determined as being present only when 100% coverage of the gene sequence is 
present. 
 
Individual genes will be reviewed and characterised by their corresponding beta-lactamase 
enzymes and distinctive substrates using the Bush-Jacoby-Medeiros classification scheme. 
Isolates will be screened for: (a) beta-lactamase genes grouped as ESBL, plasmid-mediated 
ampC, and narrow-spectrum OXA; (b) Group 2b enzymes such as TEM-1 and SHV-1; and (c) 
carbapenemase genes. 
6. Withdrawal from the PK-PD sub-study 
The patient or their substitute decision maker may withdraw consent for participation in the 
BLING III study and/or the BLING III PK-PD sub-study at any time. If consent for use of 
previously collected blood samples is withdrawn or refused, then the samples will be 
destroyed and no anlysis performed. 
7. Safety considerations 
No additional risks are anticipated with participation in the BLING III PK-PD sub-study. 
Participation in the BLING III PK-PD sub-study will involve three blood samples (3-5 mL of blood 
per sample) taken either during one dosing interval (intermittent infusion) or over an 8-hour 
period (continuous infusion). Samples will be in addition to, and may occur more frequently 
than as per standard clinical care. However, samples will be collected via existing arterial 
catheters placed as part of standard clinical care. There should be no additional discomfort 
for patients participating in the PK-PD sub-study.  

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8. Data analysis methods 
8.1. Sample size 
This is an opportunistic exploratory study with an aim to recruit 300 culture-positive sepsis 
patients in the BLING III PK-PD sub-study. Due to study design, culture negative patients will 
also be included in additional analyses. 
8.2. Statistical analysis plan 
See Appendix 1 (below). 
9. Data collection 
The PK-PD sub-study will require collection of data pertaining to the timing details of blood 
sample collection and causative organism MIC, if available. It will not require any extra data 
demographic, or outcome collection above that already specified in the BLING III Study. 
Patient demographics, randomisation allocation and outcome measures required for data 
analysis will be obtained from the routinely collected BLING III data. Plasma antibiotic 
concentration data will be supplied by the Central Bioanalysis Laboratory at UQCCR using a 
password-protected data file. 
10. Quality control and quality assurance  
All centrifuged plasma samples will be stored at -70°C to -80oC until transferred to the 
Antimicrobial Optimisation Group, UQ Centre of Clinical Research at The University of 
Queensland for assay of unbound antibiotic concentrations. Unbound concentrations of 
piperacillin and meropenem in the biological samples will be determined by chromatographic 
methods (HPLC and LC-MS/MS) that are validated and conducted in accordance with the Food 
and Drug Administration’s guidance for industry on bioanalysis. 
11. Ethical considerations 
The BLING III PK-PD sub-study will be conducted in accordance with ethical principles outlined 
as per the main BLING III study protocol (refer to section 10. ‘Ethics and dissemination’). 
The protocol, as a sub-study of the BLING III Study, will be reviewed by the relevant Human 
Research Ethics Committee/Instiutional Review Board for each participating site, and where 
possible, under a single ethical review of multicentre trial. Approval from local research 
governance will be sought as required. Documentation of approval of the protocol and 
consent documents will be provided to the coordinating centre before the study can begin at 
any site. Consent will be obtained for all patients participating in the BLING III PK-PD sub-study 
and a tick box on the BLING III consent form will indicate the patients willingness or not to 
participate in the PK-PD sub-study. 
12. Publication policy 
The BLING III PK-PD sub-study will be conducted under the name ‘BLING III PK-PD Sub-study’. 
Central project coordination and data management will be provided by the Antimicrobial 
Optimisation Group, UQCCR at the University of Queensland. 
Authorship of publications arising from the sub-study will be consistent with current ANZICS 
Clinical Trials Group policies with full credit assigned to all collaborating investigators, research 

BLING III Study: PK-PD Sub-study Protocol   Version 6.0  8th November 2022 
 
 
14 
coordinators and institutions. Responsibility for the content of manuscripts will rest with the 
BLING III PK-PD Sub-study Steering Committee. 
It is expected that findings will be disseminated via publication in high-quality peer reviewed 
journals in the medical literature. Study findings will also be presented at regional, national, 
and international intensive care conferences. 
Funding bodies will be acknowledged in all publications. 
13. Proposed project timeline 
 
The BLING III PK-PD sub-study will be conducted over a two- to three-year period with project 
milestones aligned with the main BLING III study. 
 

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14. References 
 
1. 
Craig WA. Pharmacokinetic / Pharmacodynamic Parameters : Rationale for 
Antibacterial Dosing of Mice and Men. Clinical infectious diseases 1998;26:1-12. 
2. 
Roberts Ja, Abdul-Aziz MH, Lipman J, et al. Individualised antibiotic dosing for patients 
who are critically ill: Challenges and potential solutions. The Lancet Infectious Diseases 
2014;14:498-509. 
3. 
Ambrose PG, Bhavnani SM, Rubino CM, et al. Pharmacokinetics-pharmacodynamics of 
antimicrobial therapy: it's not just for mice anymore. Clin Infect Dis 2007;44:79-86. 
4. 
Li C, Du X, Kuti JL, Nicolau DP. Clinical pharmacodynamics of meropenem in patients 
with lower respiratory tract infections. Antimicrobial agents and chemotherapy 
2007;51:1725-30. 
5. 
Tam VH, McKinnon PS, Akins RL, Rybak MJ, Drusano GL. Pharmacodynamics of 
cefepime in patients with Gram-negative infections. J Antimicrob Chemother 2002;50:425-8. 
6. 
Tam VH, Schilling AN, Neshat S, Poole K, Melnick DA, Coyle EA. Optimization of 
meropenem minimum concentration/MIC ratio to suppress in vitro resistance of 
Pseudomonas aeruginosa. Antimicrob Agents Chemother 2005;49:4920-7. 
7. 
Sinnollareddy MG, Roberts MS, Lipman J, Roberts JA. beta-lactam pharmacokinetics 
and pharmacodynamics in critically ill patients and strategies for dose optimization: a 
structured review. Clin Exp Pharmacol Physiol 2012;39:489-96. 
8. 
Mohd Hafiz AA, Staatz CE, Kirkpatrick CM, Lipman J, Roberts JA. Continuous infusion 
vs. bolus dosing: implications for beta-lactam antibiotics. Minerva Anestesiol 2012;78:94-104. 
9. 
Delattre IK, Taccone FS, Jacobs F, et al. Optimizing beta-lactams treatment in critically-
ill patients using pharmacokinetics/pharmacodynamics targets: are first conventional doses 
effective? Expert Rev Anti Infect Ther 2017;15:677-88. 
10. 
Roberts JA, Paul SK, Akova M, et al. DALI: defining antibiotic levels in intensive care 
unit patients: are current beta-lactam antibiotic doses sufficient for critically ill patients? Clin 
Infect Dis 2014;58:1072-83. 
 
 
 

BLING III Study: PK-PD Sub-study Protocol   Version 6.0  8th November 2022 
 
 
16 
15. Appendix 1 
Statistical Analysis Plan 
 
The Beta-lactam Infusion Group (BLING) III Pharmacokinetic-Pharmacodynamic (PK-PD) sub-
study is an embedded observational sub-study of the BLING III randomised controlled trial 
(RCT) conducted in patients with sepsis. This document describes the pre-specified statistical 
analysis plan finalised by the BLING III PK-PD sub-study statistician (MC) and lead 
investigators (JAR, MOC, JD and JL) and approved by the BLING III PK-PD sub-study Steering 
Committee before completion of patient enrolment and database lock. 
 
Data analysis  
The following data of PK-PD sub-study participants will be presented by treatment arm: 
baseline characteristics (Table 1), APACHE III diagnosis (Table 2), primary site of infection 
(Table 3), infective organisms identified in culture-positive patients (Table 4) and associations 
between beta-lactam antibiotic exposure and outcomes (Tables 5 and 6). 
 
Analysis for Objective 1 
According to randomisation status, a stacked bar chart showing various antibiotic exposure 
ranges will be presented for each treatment arm separately for definitions a), b) and c) (Figure 
2). 
 
Analysis for Objectives 2a) and 2b)  
Multivariable fractional polynomial logistic regression models will be used. Each model will 
include main effects for: beta-lactam antibiotic (meropenem vs. piperacillin-tazobactam, 
severity of illness [APACHE II score as a continuous variable], lung infection and non-
fermenting Gram-negative bacilli). It will be assumed that after adjustment for covariates, 
exposure will relate to outcome in a flexible manner for each infusion method (continuous vs. 
intermittent), as described below. This relationship will be shown graphically with a 95% 
confidence interval (CI) band (Figures 3a and 3b).  
 
Adjustment for extreme values:  
To limit the effect of outliers, an assumption will be made that for an unbound beta-lactam 
antibiotic concentration to MIC ratio of ≤ 0.1, outcome does not vary with exposure. The same 
assumption will be made for an unbound beta-lactam antibiotic concentration to MIC ratio of 
≥ 50. Therefore, in the analysis, exposure values < 0.1 will be replaced with 0.1 and exposure 
values > 50 will be replaced with 50 (variable name: “trimmedexposure”). A log transformed 
positive exposure variable (tr_exposure) with no extremely low values will then be created as 
follows: tr_exposure = log(trimmedexposure) - log(0.07). This exposure variable will be 
modelled, and the best second-degree fractional polynomial model selected for each infusion 
method (from the default powers in Stata v17). 
 
 
 
 

BLING III Study: PK-PD Sub-study Protocol   Version 6.0  8th November 2022 
 
 
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Additional analyses 
1. For Main Objectives 2a) and 2b), the association between beta-lactam exposure and 
outcomes will be explored within each of the following sub-groups of culture-positive 
patients#: 
a) (i) Lung infection, and (ii) non-lung infection 
b) (i) Piperacillin-tazobactam, and (ii) meropenem 
c) Severity of illness: (i) APACHE II score < 25, and (ii) APACHE II score ≥ 25[1] 
d) (i) Non-fermenting Gram-negative bacilli, and (ii) other causative organisms 
 
2. Analyses where ‘beta-lactam antibiotic exposure’ is defined as 24-hour area under the 
concentration-time curve of unbound beta-lactam antibiotic to MIC ratio (ƒAUC0-
24/MIC) will be undertaken for Main Objectives 2a) to 2c). AUC0-24 will be estimated by 
the non-parametric population pharmacokinetic software, Pmetrics.  
 
3. Analyses of culture-positive# vs. culture-negative patients^ will be undertaken for Main 
Objectives 2a) to 2c). 
 
4. Analyses for all recruited PK-PD sub-study patients (i.e. both culture-positive# and 
culture-negative patients^) will be undertaken for Main Objectives 2a) to 2c). 
  
5. Additionally, analyses will be undertaken whereby MIC values in culture-negative 
patients will be based on MIC clinical breakpoints based on Pseudomonas aeruginosa 
susceptibility (i.e. 16 mg/L for piperacillin-tazobactam and 2 mg/L for meropenem 
based on the European Committee on Antimicrobial Susceptibility Testing's MIC 
data)[2]. 
 
6. In silico resistance gene profiling to characterise the resistance gene compliment and 
resistance associated mutations in the causative organism. The association between 
predicted non-susceptibility profiles (MDR, XDR, PDR) in organisms resistant to beta-
lactam antibiotics and treatment outcomes of the main study will be explored. 
 
7. A comparison between MIC values measured using BMD and E-test® will be 
undertaken per study antibiotic. 
 
#For culture-positive patients, MIC values of causative organisms will be based on broth 
microdilution (BMD) measurements. 
 
^For culture-negative patients, median MIC values for piperacillin-tazobactam and 
meropenem based on culture-positive patient data will be used (if no organism has been 
grown, then the median MIC value among the culture-positive dataset will be used). If only 
the Gram stain is available, then the median MIC value among identified causative 
organisms with the corresponding Gram stain in the culture-positive dataset will be used; if 
species specific without a measured MIC, then the median value among corresponding 
isolated species in the same geographical location in the culture-positive dataset will be 
used, where possible. 

BLING III Study: PK-PD Sub-study Protocol   Version 6.0  8th November 2022 
 
 
18 
 
Definition of statistical significance 
A two-sided p-value < 0.05 will be considered evidence of a significant association. There will 
be no multiplicity adjustments made, although the number of analyses undertaken will be 
considered when interpreting findings. 
 
 
 

BLING III Study: PK-PD Sub-study Protocol   Version 6.0  8th November 2022 
 
 
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Proposed Figures and Tables 
 
Figure 2. Based on randomisation status, a stacked bar chart will show proportion of 
patients in various antibiotic exposure ranges based on unbound beta-lactam antibiotic 
concentrations* above the MIC (expressed as ratios):  
 
Example: 
 
 
*For patients randomised to the continuous infusion arm, the mean value of the 3 steady-
state concentrations will be used; for patients randomised to the intermittent infusion arm, 
the measured ‘trough’ concentration will be used. 
 

BLING III Study: PK-PD Sub-study Protocol   Version 6.0  8th November 2022 
 
 
20 
 
Figure 3a. Continuous exposure vs Day 90 mortality 
 
Example: 
 
 
 
Figure 3b. Continuous exposure vs failure of clinical cure at Day 14  
(A graph similar to Figure 3a. will be generated) 
 
 
 

BLING III Study: PK-PD Sub-study Protocol   Version 6.0  8th November 2022 
 
 
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Table 1. Baseline characteristics 
 
Characteristic 
Continuous Infusion 
Intermittent Infusion 
Total 
(n = XXX) 
All 
(n = XXX) 
Culture +VE 
(n = XXX) 
Culture -VE 
(n = XXX) 
All 
(n = XXX) 
Culture +VE 
(n = XXX) 
Culture -VE 
(n = XXX) 
Age (years) 
Mean ± SD  
Mean ± SD 
Mean ± SD 
Mean ± SD 
Mean ± SD 
Mean ± SD 
Mean ± SD 
Sex, female 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Weight (kg) 
Mean ± SD  
Mean ± SD 
Mean ± SD 
Mean ± SD 
Mean ± SD 
Mean ± SD 
Mean ± SD 
Height (cm) 
Mean ± SD  
Mean ± SD 
Mean ± SD 
Mean ± SD 
Mean ± SD 
Mean ± SD 
Mean ± SD 
Source of ICU 
admission 
 
 
 
 
 
 
Accident and 
Emergency 
Department 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Hospital floor 
(i.e. wards) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Transfer 
from another 
ICU 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 

BLING III Study: PK-PD Sub-study Protocol   Version 6.0  8th November 2022 
 
 
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Transfer 
from another 
hospital 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
From OT 
following 
EMERGENCY 
surgery 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
From OT 
following 
ELECTIVE 
surgery 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Time since ICU 
admission 
(hours) 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or Median 
APACHE II score 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or Median 
Lowest 
PaO2/FiO2 
ratio in the 
24 hours 
prior to 
randomisatio
n 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or Median 

BLING III Study: PK-PD Sub-study Protocol   Version 6.0  8th November 2022 
 
 
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Highest 
creatinine 
(μmol/L) 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or Median 
Highest bilirubin 
(μmol/L) 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or Median 
Lowest platelet 
count 
(x109/L) 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or Median 
Lowest MAP in 
24 hours 
prior to 
randomisatio
n (mmHg) 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or Median 
Worst Glasgow 
Coma Score 
(non-
sedated) in 
the 24 hours 
prior to 
randomisatio
n 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or 
Median 
Mean or Median 
Receiving 
inotropes/va
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 

BLING III Study: PK-PD Sub-study Protocol   Version 6.0  8th November 2022 
 
 
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sopressors in 
the 24 hours 
prior to 
randomisatio
n 
Receiving 
antibiotics in 
the 24 hours 
prior to 
randomisatio
n1 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
1Other than piperacillin-tazobactam or meropenem. 
APACHE = Acute Physiology and Chronic Health Evaluation. ICU = intensive care unit. MAP = mean arterial pressure. OT = operating theatre. SD = standard deviation. 
 
 
 

BLING III Study: PK-PD Sub-study Protocol   Version 6.0  8th November 2022 
 
 
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Table 2. APACHE III diagnosis 
 
Diagnosis 
Continuous Infusion 
Intermittent Infusion 
Total 
(n = XXX) 
All 
(n = XXX) 
Culture +VE 
(n = XXX) 
Culture -VE 
(n = XXX) 
All 
(n = XXX) 
Culture +VE 
(n = XXX) 
Culture -VE 
(n = XXX) 
   Non-operative diagnoses 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Cardiovascular 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Respiratory 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Gastrointestinal 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Neurological 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Sepsis 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Trauma 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Metabolic 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Haematological  
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Renal/genitourinary  
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Musculoskeletal/skin 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Other 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 

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Operative diagnoses 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Cardiovascular 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Respiratory 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Gastrointestinal 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Neurological 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Trauma 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Metabolic 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Haematological  
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Renal/genitourinary  
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Gynaecological 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Musculoskeletal/skin  
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
 
 
 

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Table 3. Primary site of infection 
 
Primary site of infection 
Continuous Infusion 
Intermittent Infusion 
Total 
(n = XXX) 
All 
(n = XXX) 
Culture +VE 
(n = XXX) 
Culture -VE 
(n = XXX) 
All 
(n = XXX) 
Culture +VE 
(n = XXX) 
Culture -VE 
(n = XXX) 
Pulmonary 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Intra-abdominal 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Blood 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Skin 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Urinary 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Intravenous catheter 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Central nervous system 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Gut  
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Endocarditis  
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Other 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
 

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Table 4. Infective organisms identified in culture-positive patients 
 
Organism 
Continuous Infusion 
(n = XXX) 
Intermittent Infusion 
(n = XXX) 
Total 
Gram positive bacteria  
n (%) 
n (%) 
n (%) 
Methicillin-susceptible  
Staphylococcus aureus 
n (%) 
n (%) 
n (%) 
Methicillin-Resistant 
Staphylococcus aureus  
n (%) 
n (%) 
n (%) 
Streptococcus pneumoniae  
n (%) 
n (%) 
n (%) 
Beta-haemolytic streptococci 
(Group A, B or C)  
n (%) 
n (%) 
n (%) 
Viridans group Streptococci  
n (%) 
n (%) 
n (%) 
Enterococcus species  
n (%) 
n (%) 
n (%) 
Coagulase-negative staphylococci  
n (%) 
n (%) 
n (%) 
Nocardia species 
n (%) 
n (%) 
n (%) 
Gram negative bacteria  
n (%) 
n (%) 
n (%) 
Pseudomonas species  
n (%) 
n (%) 
n (%) 

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Burkholderia species  
n (%) 
n (%) 
n (%) 
Haemophilus species  
n (%) 
n (%) 
n (%) 
Acinetobacter species  
n (%) 
n (%) 
n (%) 
Klebsiella pneumoniae 
n (%) 
n (%) 
n (%) 
Klebsiella aerogenes 
n (%) 
n (%) 
n (%) 
Enterobacter species  
n (%) 
n (%) 
n (%) 
Escherichia species  
n (%) 
n (%) 
n (%) 
Serratia species  
n (%) 
n (%) 
n (%) 
Bacteroides species 
n (%) 
n (%) 
n (%) 
Neisseria meningitidis  
n (%) 
n (%) 
n (%) 
Neisseria gonorrhoeae 
n (%) 
n (%) 
n (%) 
Campylobacter species  
n (%) 
n (%) 
n (%) 
Citrobacter species  
n (%) 
n (%) 
n (%) 
Proteus species  
n (%) 
n (%) 
n (%) 
Stenotrophomonas maltophilia 
n (%) 
n (%) 
n (%) 
Other 
n (%) 
n (%) 
n (%) 

BLING III Study: PK-PD Sub-study Protocol   Version 6.0  8th November 2022 
 
 
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Mycobacterium species 
n (%) 
n (%) 
n (%) 
Mixed anaerobes  
n (%) 
n (%) 
n (%) 
Other 
n (%) 
n (%) 
n (%) 
As there may be more than 1 infective organism per participant, percentages may not add up to 100%. 
 

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31
Table 5. Unadjusted associations between beta-lactam antibiotic exposure ranges (based on unbound concentrations* above the MIC) and 
patient outcomes in culture-positive patients 
Outcome and 
group 
Beta-lactam antibiotic exposure 
<0.1 
0.1 to 0.5 
0.5 to 1 
1 to 4 
4 to 20 
20 to 50 
>50 
All-cause patient 
mortality at Day 
90  
 
 
 
 
 
 
 
Continuous 
Infusion 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
Intermittent 
Infusion 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
Total 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
Failure of clinical 
cure at Day 14 
 
 
 
 
 
 
 
Continuous 
Infusion 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
Intermittent 
Infusion 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
Total 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
*For patients randomised to the continuous infusion arm, the mean value of the 3 steady-state concentrations will be used; for patients 
randomised to the intermittent infusion arm, the measured ‘trough’ concentration will be used. 
 

BLING III Study: PK-PD Sub-study Protocol   Version 6.0  8th November 2022 
 
 
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Table 6. Unadjusted associations between beta-lactam antibiotic exposure ranges (based on unbound concentrations* above the MIC) and 
antimicrobial resistance outcomes in culture-positive patients 
 
Outcome and group 
Beta-lactam antibiotic exposure 
<0.1 
0.1 to 0.5 
0.5 to 1 
1 to 4 
4 to 20 
20 to 50 
>50 
New acquisition, 
colonisation or 
infection with a multi-
resistant organism or 
Clostridium difficile 
associated diarrhoea 
up to Day 14 
 
 
 
 
 
 
 
Continuous Infusion 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
Intermittent Infusion 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
Total 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
n/N (XX %) 
 
*For patients randomised to the continuous infusion arm, the mean value of the 3 steady-state concentrations will be used; for patients 
randomised to the intermittent infusion arm, the measured ‘trough’ concentration will be used.

References 
 
1.  
Billot L, Lipman J, Brett SJ, De Waele JJ, Cotta MO, Davis JS, Finfer S, Hammond N, 
Knowles S, McGuinness S et al: Statistical analysis plan for the BLING III study: a phase 
3 multicentre randomised controlled trial of continuous versus intermittent beta-
lactam antibiotic infusion in critically ill patients with sepsis. Critical Care and 
Resuscitation 2021, 23(3):273-284. 
2. 
European Committee on Antimicrobial Susceptibility Testing Steering C: EUCAST 
Technical Note on linezolid. Clin Microbiol Infect 2006, 12(12):1243-1245.