Tobias Bäckström

Georgios Panagiotidis

Olof Beck

Charlotte Asker-Hagelberg

Mamun-Ur Rashid

Andrej Weintraub

Carl Erik Nord
email: carl.erik.nord@ki.se

Effect of ceftobiprole on the normal human intestinal microflora

Keywords:

and six females) aged 20-31 years received ceftobiprole 500 mg by intravenous infusion every 8 h for 7 days. Plasma samples were collected on Days -1, 1, 4, 7, 10, 14 and 21 for determination of drug concentration by biological and chemical methods. Faecal samples were collected on Days -1, 2, 4, 7, 10, 14 and 21. For analysis of the microflora, faecal specimens were cultured on non-selective and selective media. Different colony types were counted, isolated in pure culture and identified to genus level. All new colonising aerobic and anaerobic bacteria were tested for susceptibility to ceftobiprole. Plasma concentrations of ceftobiprole 10 min after completion of infusion were as follows: Day 1, 14.7-23.6 mg/L; Day 4, 15.9-24.5 mg/L; and Day 7, 15.9-23.9 mg/L. No ceftobiprole was detected in plasma on Days -1, 10, 14 and 21. No measurable concentrations of ceftobiprole were found in faeces on Days -1, 2, 4, 7, 10, 14 and 21. There were minor changes in the numbers of enteric bacteria, enterococci and Candida albicans and there were moderate changes in the numbers of bifidobacteria, lactobacilli, clostridia and Bacteroides spp.

during the same period. No Clostridium difficile strains or toxins were found. No new colonising aerobic and anaerobic bacteria with ceftobiprole minimum inhibitory concentrations of 4 mg/L were found. Ceftobiprole had no significant ecological impact on the human intestinal microflora. 4 efficacy in patients with infections due to MRSA and is expected to be a useful addition to the armamentarium of agents for the treatment of complicated skin infections and pneumonia [START_REF] Barbour | Ceftobiprole: a novel cephalosporin with activity against Gram-positive and Gram-negative pathogens, including methicillin-resistant Staphylococcus aureus (MRSA)[END_REF].

The purpose of the present study was to investigate the effect of ceftobiprole on the intestinal microflora over 7 days of administration. Careful investigation of the effect of antibiotic treatment on the endogenous microflora is of importance since alteration of the endogenous flora balance, qualitatively and/or quantitatively, may facilitate colonisation by new potentially pathogenic strains or enable microorganisms already present in the normal flora to develop resistance. Pharmacokinetics and pharmacodynamics are further important consideration for the safety and efficacy of antimicrobial agents [START_REF]The European Agency for the Evaluation of Medicinal Products, Committee for Proprietary Medicinal Products (CPMP). Points to consider on pharmacokinetics and pharmacodynamics in the development of antibacterial medicinal products[END_REF].

Materials and methods

Objectives

The primary objective was to assess the effect of antibiotic treatment on the intestinal microflora before, during and after administration of ceftobiprole given to healthy

Study design

This was an open-label, non-comparative, multiple-dose, single-centre study in 12 healthy subjects. Each subject received 500 mg of ceftobiprole by intravenous (i.v.) infusion over 120 min every 8 h (q8h) for 7 days. Subjects were admitted to the study centre the day before the first drug administration and were discharged from the study centre on Day 8. Subjects visited the study centre for follow-up visits on Days 10, 14 and 21. A total of 13 plasma samples were collected from each subject, including 1 at pre dose (Day -1), 3 samples each on Days 1, 4 and 7 and 1 sample each on Days 10, 14 and 21. A total of seven faecal samples were collected, including at pre-dose (Day -1) and on Days 2, 4, 7, 10, 14 and 21. Each subject included in the study participated at follow-up visits on Days 10, 14 and 21. Additional safety evaluations included physical examination, vital signs, clinical laboratory tests, 12-lead electrocardiogram (ECG) and adverse event monitoring.

Subjects

Twelve healthy subjects (six males and six females) aged 20-31 years were included in the study. They were recruited through information about the study on the Clinical Pharmacology Trial Unit website on the internet. Physical examination was carried out on each subject before the start of the study, including measurements of blood pressure and heart rate, ECG and clinical laboratory safety tests as well as an interview on medical and surgical history. Female subjects were tested for history of alcohol abuse or drug abuse; and donation of blood or blood products within 1 month preceding the study. Before being admitted to the study, subjects gave consent to participate after the nature, scope and possible consequences of the study had been explained in a form understandable to him or her. The subject was also given written information and a copy of the signed consent form.

Approvals

The study protocol submitted to the Ethics Committee of Karolinska Institutet (Stockholm, Sweden) and to the Medical Products Agency (Uppsala, Sweden) was approved before starting the trial.

Drug administration

Subjects each received ceftobiprole 500 mg by i.v. infusion over 120 min q8h for 7 days. Samples were obtained for bioassay and chemical analysis of ceftobiprole from faecal specimens passed on study Days -1, 2, 4, 7, 10, 14 and 21, unless no specimen was passed on a given day, in which instance the first subsequent specimen passed after that day, before the next collection time point, was collected.

If more than one specimen was produced on a specified day, only the first specimen on that day was collected.

Plasma and faecal samples for the analyses were collected in sterile tubes and containers and were frozen at -70 C until processed. ATCC 9341 as indicator strain [START_REF] Panagiotidis | Effect of ceftaroline on normal human intestinal microflora[END_REF]. Two validation reports were provided by the investigator giving information on linearity ranges, lower limit of detection as well as intra-assay and inter-assay coefficient of variation. Standards were prepared in faeces for faecal samples.

Determination of ceftobiprole plasma and faecal concentrations

Processing of faecal specimens for microbiological analyses

Samples were obtained for culture and ceftobiprole susceptibility tests from faecal specimens passed on study Days -1, 2, 4, 7, 10, 14 and 21, unless no specimen was passed on a given day in which instance the first subsequent specimen passed was collected. Samples were suspended in pre-reduced peptone yeast extract medium, A c c e p t e d M a n u s c r i p t 9 diluted ten-fold and inoculated on non-selective and selective agars as described by Nord et al. [START_REF] Nord | Effect of dalbavancin on the normal intestinal microflora[END_REF]. Aerobic agar plates were incubated for 24 h at 37 C and anaerobic plates were incubated for 48 h at 37 C in anaerobic jars (BBL GasPak TM ; BD, Cockeysville, MD). Following incubation, different colony types were counted, isolated in pure culture and identified to genus level. All isolates were analysed according to Gram-stain appearance and colony morphology followed by biochemical tests [START_REF] Murray | Manual of clinical microbiology[END_REF]. Anaerobic microorganisms were identified by gas chromatographic analysis of metabolites from glucose. Clostridium difficile strains were further characterised by cytotoxicity assay, polymerase chain reaction (PCR) ribotyping and a multiplex realtime PCR (Xpert TM C. difficile; Cepheid, San Francisco, CA) as described recently [START_REF] Huang | Comparison of a commercial multiplex real-time PCR to the cell cytotoxicity neutralization assay for diagnosis of Clostridium difficile infections[END_REF].

The lower limit of detection for faeces was 10 2 colony-forming units (CFU)/g.

Ceftobiprole susceptibility tests

MICs for ceftobiprole were determined for new colonising strains from the faecal samples by the agar dilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines [START_REF]Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically[END_REF][START_REF]Methods for antimicrobial susceptibility testing of anaerobic bacteria; approved standard[END_REF]. The final inoculum was 10 4 CFU/spot for aerobic bacteria and 10 5 CFU/spot for anaerobic bacteria. Inoculated plates were incubated for 24 h (aerobic bacteria) and 48 h (anaerobic bacteria). Reference strains were S.

Statistical methods

Statistics were calculated for the values estimated for the faecal specimens log number of microorganisms per gram faeces and faecal concentrations of ceftobiprole as well as for the pharmacokinetic analysis by use of Wilcoxon signed-rank test.

Results

Ceftobiprole concentrations in plasma and faeces

Plasma ceftobiprole concentrations are shown in Table 1. Concentrations in samples taken 10 min after completion of infusion were as follows: Day 1, 14.7-23.6 mg/L (mean 19.4 mg/L); Day 4, 15.9-24.5 mg/L (mean 20.5 mg/L); and Day 7, 15.9-23.9 mg/L (mean 20.3 mg/L). No ceftobiprole was detected in plasma on Days -1, 10, 14 and 21. No measurable faecal concentrations were found on Days -1, 2, 4, 7, 10, 14 and 21.

A c c e p t e d M a n u s c r i p t 11

Effect of ceftobiprole on the aerobic intestinal microflora

The effect of ceftobiprole on the aerobic intestinal microflora is shown in Fig. and then increased 2 log CFU/g of faeces to Day 14; on Day 21 the numbers of enterococci were recovered to baseline. The numbers of Candida albicans were within the normal variation. Changes in the aerobic intestinal microflora (2 log CFU/g of faeces) were not significant.

Effect of ceftobiprole on the anaerobic intestinal microflora

The effect of ceftobiprole on the anaerobic intestinal microflora is shown in Fig. 2.

There were no changes in the numbers of lactobacilli and bifidobacteria from Day -1 to Day 21. Counts of clostridia increased from Day 2 to Day 7 by ca. 1.5 log CFU/g of faeces and then returned to baseline counts. The numbers of Bacteroides were only influenced on Day 2, with a decrease of ca. 0.5 log CFU/g of faeces. All alterations were within the normal variation and significance. No C. difficile strains or toxins A and B were found.

Ceftobiprole susceptibility tests

No new colonising aerobic and anaerobic bacteria resistant to ceftobiprole (MIC  4 mg/L) were found.

. Safety and tolerability

There were 10 subjects with 28 adverse events, all mild in severity. Four events of common cold, one event of very mild swelling of the lower lip, one event of vasovagal reaction in relation to i.v. cannula insertion and one event of muscle pain were deemed to be unrelated to the study drug. Other adverse events were deemed to be possibly related to ceftobiprole and included infusion-site reactions in three subjects, with pain, mild swelling and thrombophlebitis in one subject. Mild rash was seen in three subjects and vaginal Candida infection in two subjects. Two subjects had two events of headache each. One subject had two events of mild diarrhoea. Nausea was seen in one subject. During study drug infusion, five subjects noticed mild taste alterations. No subjects had potentially clinically significant changes in post-baseline vital sign values. No subjects had potentially clinically significant changes in postbaseline haematology, chemistry or urine analysis based both on normal ranges and on percent change from baseline. There were no significant changes in ECG parameters from baseline to post baseline in any subject.

Discussion

Ceftobiprole is active against most aerobic Gram-positive bacteria as well as against many Enterobacteriaceae and Pseudomonas aeruginosa strains. Anaerobic Grampositive bacteria such as bifidobacteria, propionibacteria and peptostreptococci are susceptible, whilst clostridia are variable in susceptibility to ceftobiprole. The MIC for C. difficile strains is between 1.0 mg/L and 8.0 mg/L. Bacteroides fragilis and Prevotella spp. are resistant to ceftobiprole [START_REF] Ednie | Antianaerobe activity of ceftobiprole: a new broad-spectrum cephalosporin[END_REF].

A c c e p t e d M a n u s c r i p t 13 Ceftobiprole is a promising antimicrobial agent for treatment of complicated skin infections and pneumonia. Ceftobiprole is reported to be well tolerated with good safety, which was also observed in the present investigation [START_REF] Schirmer | Ceftobiprole: a new cephalosporin for the treatment of skin and skin structure infections[END_REF].

Administration of antibacterial agents can cause disturbances in the ecological balance between the host and microorganisms. These changes are dependent on the spectrum of activity, dose, route of administration, pharmacokinetic and pharmacodynamic properties and in vitro inactivation of the agent. Secretion of an agent by intestinal mucosa or bile may have an impact on the intestinal microflora leading to antibiotic resistance. The effect of ceftobiprole on the normal intestinal microflora has not been studied previously. No faecal concentration of ceftobiprole was found and the minor effect on the intestinal microflora is thus explained by these findings. Ceftobiprole is mainly eliminated by renal excretion [START_REF] Murthy | Pharmacokinetics and pharmacodynamics of ceftobiprole, an anti-MRSA cephalosporin with broadspectrum activity[END_REF]. A c c e p t e d M a n u s c r i p t

A c c e p t e d M a n u s c r i p t 2 ABSTRACT

 2 Ceftobiprole is a new broad-spectrum pyrrolidinone cephem active against meticillinresistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis and Gram-negative bacteria such as Enterobacteriaceae and Pseudomonas spp. The purpose of the present study was to investigate the effect of administration of ceftobiprole on the normal intestinal microflora. Twelve healthy subjects (six males

  Under usual circumstances, the normal human microflora is relatively stable in each ecological habitat and acts as a barrier against colonisation by potentially pathogenic microorganisms. Disturbances in the normal microflora may occur due to changes in diet, radiation or administration of antimicrobial agents. Administration of antimicrobial agents can cause several adverse effects on the microflora. Emergence of resistance among bacteria in the normal flora and distribution of resistance genes by transfer of DNA in the microbial community can contribute to an increased load of resistant, potentially pathogenic microorganisms. Another effect of a disturbed normal microflora is reduction in colonisation leading to overgrowth of already present microorganisms or of exogenic pathogens[START_REF] Sullivan | Interaction between antimicrobial agents and the oropharyngeal and intestinal microflora[END_REF]. The extent of disturbances is influenced by the spectrum of the agent, dose, route of administration, pharmacokinetic and pharmacodynamic properties and in vivo inactivation of the agent. Parenterally administered agents that are excreted in the bile or the intestinal mucosa may interfere with the normal intestinal microflora[START_REF] Sullivan | Effect of antimicrobial agents on the ecological balance of human microflora[END_REF].Increased resistance in manyGram-positive bacteria has driven the need to develop new antibacterial agents with activity against these pathogens, including meticillinresistant Staphylococcus aureus (MRSA). Ceftobiprole is a new broad-spectrum pyrrolidinone cephem active against Gram-positive bacteria including S. aureus (MRSA and meticillin-susceptible), vancomycin-resistant Enterococcus faecalis and Gram-negative bacteria such as Enterobacteriaceae and Pseudomonas spp. Ceftobiprole has undergone extensive evaluation in phase I studies to characterise dose, pharmacokinetics and safety/tolerability as well as in an early phase II study and subsequent phase III studies. It is the first cephalosporin to demonstrate clinical

  individuals. Secondary objectives were: (i) to explore the potential for development of resistance by measuring the susceptibility [minimum inhibitory concentrations (MICs)] of new colonising isolated bacterial strains before, during and after antibiotic treatment; (ii) to correlate intestinal microflora patterns with ceftobiprole concentrations measured in

A c c e p t e d M a n u s c r i p t 5 plasma

 5 and faeces; and (iii) to investigate the concentration of ceftobiprole in faeces compared with plasma.

  criteria were men and women aged between 18 years and 45 years and normal findings in the medical history and physical examination. Exclusion criteria were: regular use of medication except contraceptive tablets; treatment with antimicrobial agents within the 3 months preceding the study; participation in a trial with another investigational drug within 1 month preceding the study; present or residual gastrointestinal, liver or kidney disease, or other conditions known to interfere with the absorption, distribution, metabolism or excretion of drugs; known history of Clostridium difficile infection; history of hypersensitivity to -lactam antibiotics; pregnant or breast-feeding women; estimated creatinine clearance of <80 mL/min; positive screen for hepatitis B or C or human immunodeficiency virus (HIV);

  A c c e p t e d M a n u s c r i p t 7 2.6. Sampling of specimens Plasma samples were obtained for chemical analysis of ceftobiprole on study Day -1, after the start of the first ceftobiprole infusion on study Days 1, 4 and 7 and on study Days 10, 14 and 21.

  Plasma and faecal concentrations of ceftobiprole were determined by the following developed and validated methods by liquid chromatography-tandem mass spectrometry. Concentrations of ceftobiprole in Li-heparin human plasma (pH 5) were determined using an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) system (Waters Corp., Milford, MA) following sample preparation by protein precipitation with 1% formic acid in acetonitrile containing BAL0009141-d4 as an internal standard. The final extract of plasma sample was A c c e p t e d M a n u s c r i p t 8 injected into the UPLC-MS/MS system. Concentrations of ceftobiprole in faecal extract (supernatant from human faecal acidic homogenised with phosphate-buffered saline solution) were determined following sample preparation by precipitation with 1% formic acid in acetonitrile containing BAL0009141-d4 as an internal standard. The supernatant was diluted with the same volume of 1% formic acid and injected into the UPLC-MS/MS system. Separation of the analytes was achieved on an ACQUITY UPLC BEH C 18 column (1.7 m, 2.1  50 mm) (Waters Corp.) using a gradient run with mobile phase A of 0.1% formic acid in water and mobile phase B of acetonitrile. Analytes were detected using a Micromass Quattro Premier XE mass spectrometer (Waters Corp.) operating in positive electrospray ionisation mode utilising multiple reaction monitoring for the transitions 535203 m/z for ceftobiprole and 539207 m/z for IS (International System of Units). The calibration curve in plasma was linear over the range 0.05-30 mg/L and in faecal extract over the range 1.00-143 mg/kg faeces. Ceftobiprole concentrations in faeces were also biologically determined in Antibiotic Medium 1 (Difco, Sparks, MD) with Micrococcus luteus

aureus

  ATCC 29213, E. faecalis ATCC 29212, Escherichia coli ATCC 25922, Bacteroides fragilis ATCC 25285 and C. difficile ATCC 700057. Isolated strains were considered resistant according to CLSI breakpoints (recommended breakpoint for ceftobiprole is 4 mg/L). The MIC was defined as the lowest concentration of drug that inhibited growth completely. MIC 50 and MIC 90 values corresponded to the concentrations that inhibited the growth of 50% and 90% of the strains tested, respectively.

  The following safety assessments were performed: physical examination on study Days -1, 1, 4, 7 and 10 as well as follow-up visit (Day 14) and end-of-study visit (Day 21); vital signs; ECG (Days -1 and 21); adverse events monitoring; and monitoring of clinical laboratory parameters.

  1. Mean counts of E. coli decreased by ca. 1.5 log CFU/g of faeces from study Day -1 to study Day 7, with recovery to baseline counts on Day 21. Mean values for Enterobacteriaceae did not change from study Day -1 to study Day 21. Mean numbers of enterococci decreased 1.0 log CFU/g of faeces from Day -1 to Day 7

  No new colonising ceftobiprole-resistant aerobic and anaerobic bacteria in the normal intestinal flora were recovered, probably owing to less selective pressure for the emergence of colonisation by resistant microorganisms. Clostridium difficile infection (CDI) is an adverse event mainly associated with antibiotic treatment and prophylaxis. Broadspectrum antibiotics such as cephalosporins, fluoroquinolones and amoxicillin are most often involved in causing this infection. In the present trial, no clinical C. difficile infection was observed, probably due to no biological activity of ceftobiprole in the intestine. Based on these findings, ceftobiprole has a favourable ecological profile. However, when ceftobiprole is used in hospitalised patients with serious infections and pre-existing C. difficile strains, the risk of development of CDI should be monitored.

Fig. 1 .

 1 Fig. 1. Effect of ceftobiprole administration on the aerobic intestinal microflora of 12

Fig. 2 .

 2 Fig. 2. Effect of ceftobiprole administration on the anaerobic intestinal microflora of

Table 1

 1 Ceftobiprole plasma concentrations 10 min after completion of infusion in 12 subjectsreceiving 500 mg doses of ceftobiprole intravenously every 8 h for 7 days

	Subject Ceftobiprole concentration (mg/L)
		Day -1 Day 1	Day 4	Day 7	Day 10 Day 14 Day 21
	1	N/D	19.5	22.0	18.0	N/D	N/D	N/D
	2	N/D	14.7	17.3	15.9	N/D	N/D	N/D
	3	N/D	20.4	23.1	20.7	N/D	N/D	N/D
	4	N/D	15.6	21.8	20.8	N/D	N/D	N/D
	5	N/D	22.9	23.1	21.9	N/D	N/D	N/D
	6	N/D	15.3	16.9	20.1	N/D	N/D	N/D
	7	N/D	20.9	18.7	19.9	N/D	N/D	N/D
	8	N/D	22.5	21.3	21.7	N/D	N/D	N/D
	9	N/D	21.4	22.7	21.9	N/D	N/D	N/D
	10	N/D	18.1	15.9	18.6	N/D	N/D	N/D
	11	N/D	23.6	24.5	23.9	N/D	N/D	N/D
	12	N/D	18.1	18.5	19.7	N/D	N/D	N/D
	Range -	14.7-23.6 15.9-24.5 15.9-23.9 -	-	-
	Mean	-	19.4	20.5	20.3	-	-	-
	N/D, not detected.

Edited Table

1

Funding

This study was funded by Janssen-Cilag Medical Affairs EMEA, a division of Janssen Pharmaceutica N.V.

Competing interests

None declared.

Ethical approval

The study protocol was approved by the Ethics Committee of Karolinska Institute (Stockholm, Sweden) and the Medical Products Agency (Uppsala, Sweden).


K J Christiansen

M Ip

H B Ker

M Mendoza

L Hsu

P Kiratisin

A Chongthaleong

I S Redjeki

A Quintana

R Flamm

J Garcia

M Cassettari

D Cooper

P Okolo

I Morrissey
email: ian.morrissey@quotientbioresearch.com

In vitro activity of doripenem and other carbapenems against contemporary Gram-negative pathogens isolated from hospitalised patients in the Asia-Pacific region: results of the COMPACT Asia-Pacific

Keywords:

In vitro activity of doripenem and other carbapenems against contemporary

Gram-negative pathogens isolated from hospitalised patients in the Asia-Pacific region: results of the COMPACT Asia-Pacific Study

determination against these pathogens. Data from eight countries within the Asia-Pacific region, which collected 1612 bacterial isolates, are presented here. Etest methodology was found to be a reliable method for MIC determination. Doripenem showed in vitro activity similar to or better than meropenem and at least four-fold better than imipenem against Enterobacteriaceae. Against Pseudomonas aeruginosa, doripenem was also the most active of the three carbapenems in vitro.

However, in vitro results do not necessary correlate with clinical outcome.

A c c e p t e d M a n u s c r i p t

Introduction

Antibiotic resistance amongst Gram-negative nosocomial pathogens continues to increase and to compromise treatment outcomes in the Asia-Pacific region [START_REF] Hsin-Chun | Clinical and economic impact of multidrug resistance in nosocomial Acinetobacter baumannii bacteremia[END_REF][START_REF] Prashanth | Nosocomial infections due Acinetobacter species: clinical findings, risk and prognostic factors[END_REF]. The emergence of Acinetobacter baumannii as a cause of hospital-acquired pneumonia, increasing rates of infections caused by extended-spectrum -lactamase (ESBL)producing Enterobacteriaceae such as Klebsiella pneumoniae, and the increasing resistance seen in Pseudomonas aeruginosa are just three examples [START_REF] Slama | Gram-negative antibiotic resistance: there is a price to pay[END_REF].

Antibiotic resistance rates in some countries in the Asia-Pacific Rim are considerably higher than those in other parts of the world [START_REF] Bouchillon | Comparative in vitro activity of tigecycline and other antimicrobials against Gram-negative and Grampositive organisms collected from the Asia-Pacific Rim as part of the Tigecycline Evaluation and Surveillance Trial (TEST)[END_REF], e.g. 52% of Escherichia coli (ranging from 2.6% in Australia to 83% in India) and 52% of K. pneumoniae (ranging from 7.1% in Australia to 84% in India) were found to be ESBL-producers [START_REF] Bell | Doripenem activity against leading bacterial pathogens in the Asia-Pacific region: report from the SENTRY Antimicrobial Surveillance Program[END_REF].

Infections caused by multidrug-resistant (MDR) Gram-negative pathogens have been associated with increased mortality, increased hospital stay and increased costs [START_REF] Harris | Epidemiology and clinical outcomes of patient with multiresistant Pseudomonas aeruginosa[END_REF][START_REF] Tacconelli | Multidrug-resistant Pseudomonas aeruginosa bloodstream infections: analysis of trends in prevalence and epidemiology[END_REF][START_REF] Schwaber | Clinical and economic impact of bacteremia with extended-spectrum--lactamaseproducing Enterobacteriaceae[END_REF][START_REF] Kwon | Impact of imipenem resistance on mortality in patients with Acinetobacter bacteremia[END_REF]. Furthermore, emergence of resistance during treatment of infections caused by pathogens such as P. aeruginosa and Enterobacter spp. has a dramatic effect both on outcome and costs [START_REF] Carmeli | Health and economic outcomes of antibiotic resistance in Pseudomonas aeruginosa[END_REF][START_REF] Cosgrove | Health and economic outcomes of the emergence of third-generation cephalosporin resistance in Enterobacter species[END_REF].

Carbapenems now play a prominent therapeutic role, especially in hospitals and patient populations where MDR strains have become prevalent, due to their broad spectrum of activity and their activity against pathogens such as ESBL-producing Enterobacteriaceae, P. aeruginosa and Acinetobacter spp. [START_REF] Slama | Gram-negative antibiotic resistance: there is a price to pay[END_REF][START_REF] Jones | Doripenem (S-4661), a novel carbapenem: comparative activity against contemporary pathogens including bactericidal action and preliminary in vitro methods evaluations[END_REF][START_REF] Nicolau | Carbapenems: a potent class of antibiotics[END_REF]. Doripenem is a new, broad-spectrum, intravenous carbapenem antibiotic. Its in vitro antibacterial spectrum includes meticillin-susceptible staphylococci, streptococci (including The recommended dose of doripenem (500 mg) is administered as a 1-h infusion three times a day. However, in seriously ill patients (e.g. those with VAP), 500 mg three times a day given as a 4-h infusion is recommended to maximise therapeutic benefit [START_REF] Chastre | Efficacy and safety of intravenous infusion of doripenem versus imipenem in ventilatorassociated pneumonia: a multicenter, randomized study[END_REF]. Pharmacodynamic modelling suggests that prolonged infusion (over 4 h), thus increasing the time interval where drug levels exceed the minimum inhibitory concentration (MIC), would improve coverage of pathogens having a high doripenem MIC (4 mg/L) [START_REF] Bhavnani | Use of pharmacokinetic-pharmacodynamic target attainment analyses to support phase 2 and 3 dosing strategies for doripenem[END_REF]. Clinical efficacy in patients known or suspected to be infected with pathogens having a high doripenem MIC needs to be confirmed in future clinical studies. Appropriate doripenem dose administration requires adequate epidemiological data and it is therefore essential to collect data on carbapenem MIC distribution against relevant species.

The Comparative Activity of Carbapenems Testing (COMPACT) Study had two objectives: first, to compare the MIC distribution of doripenem with that of imipenem A c c e p t e d M a n u s c r i p t 5 and meropenem against Gram-negative pathogens causing serious infections in hospitalised patients, with a particular emphasis on P. aeruginosa; and, second, to assess the reliability of Etest methodology for the determination of doripenem MICs for these pathogens. Here we report the findings from the Asia-Pacific region, including Australia, Hong Kong, India, Indonesia, Malaysia, Philippines, Singapore and Thailand.

Materials and methods

Pathogens

The COMPACT Asia-Pacific Study Group consisted of 27 medical centres in eight countries, including Australia (3 centres), Hong Kong (2 centres), India (3 centres), Indonesia (1 centre), Malaysia (3 centres), Philippines (3 centres), Singapore (2 centres) and Thailand (10 centres). Participating centres collected a protocol-defined set and number of consecutive Gram-negative isolates during the latter half of 2008 and early 2009. Non-duplicate isolates were obtained from patients with cIAIs, bloodstream infections (BSIs) or NP (including VAP). The target number of isolates to be collected at each centre was 30 P. aeruginosa, 24 Enterobacteriaceae and 6 other Gram-negative pathogens.

In vitro investigations

Each collecting centre determined the MIC of doripenem, imipenem and meropenem against each pathogen collected using Etest methodology (AB BIODISK, Solna, Sweden) following the manufacturer's guidelines. Each centre, with the exception of the centre in Indonesia that was not permitted to export isolates out of the country, A c c e p t e d M a n u s c r i p t 6 sent all isolates collected to the central reference laboratory (Quotient Bioresearch Ltd., Fordham, UK) for confirmation of pathogen identification. For quality control purposes, and for validation of Etest methodology for doripenem susceptibility testing, the central reference laboratory determined the MIC of doripenem, imipenem and meropenem by Etest and by broth microdilution (BMD) [START_REF]Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically[END_REF] against all isolates deemed to be resistant to either meropenem or imipenem or non-susceptible to doripenem according to the collecting centre's Etest result interpreted using current Clinical and Laboratory Standards Institute (CLSI) breakpoints for imipenem and meropenem (Table 1) [START_REF]Performance standards for antimicrobial susceptibility testing. Nineteenth informational supplement[END_REF] and US Food and Drug Administration (FDA) breakpoints for doripenem (Table 1) as there are no CLSI breakpoints. Further explanation on the rationale for the choice of breakpoints is included in the 'Discussion'. In addition, carbapenem susceptibility by Etest and BMD of a randomly selected 10% of susceptible isolates was determined. Quality control isolates used were H. influenzae ATCC 49766, E. coli ATCC 25922 and P. aeruginosa ATCC 27853. All data were collated and analysed by Quotient Bioresearch Ltd.

Results

Isolates collected

A total of 1612 isolates were collected over a 6-month period, consisting of 656 Enterobacteriaceae (40.7%), 784 Pseudomonas spp. (48.6%) and 172 other Gramnegative isolates (10.7%). Escherichia coli (38.6%), K. pneumoniae (35.4%) and Enterobacter spp. (10.4%) were the most predominant Enterobacteriaceae collected.

Of the Enterobacter spp. isolated, 72.1% were Enterobacter cloacae. Pseudomonas aeruginosa (99.5%) was the most commonly isolated Pseudomonas spp., and A. Pathogens were most commonly isolated from patients with NP (49.7%), followed by patients with BSIs (36.6%) and patients with cIAIs (13.7%). Pathogens were isolated from patients in the Intensive Care Unit (ICU) in 38.5% of cases.

Etest minimum inhibitory concentration determination

The reference laboratory determined the BMD and Etest MICs of 454 isolates, consisting of 306 isolates deemed to be non-susceptible to doripenem or resistant to meropenem or imipenem according to the Etest MIC results (Table 1) generated at each collecting centre as well as for 148 randomly selected isolates that were susceptible to all three carbapenems.

The MIC determined by BMD and by Etest at the central reference laboratory was the same or within one doubling dilution for 89.6% of isolates tested and was the same or within two doubling dilutions for 98% of isolates.

The MIC determined by Etest at each centre and at the central reference laboratory was the same or within one doubling dilution for 83.2% of isolates and the same or within two doubling dilutions for 92.3% of isolates. Centre Etest MIC values were higher than reference laboratory values in 26.5% of cases and lower in 20.9%.

Enterobacteriaceae

Doripenem and meropenem had activity against all Enterobacteriaceae, with MIC 90 values (MIC for 90% of the organisms) of 0.06 mg/L and 0.12 mg/L, respectively, both being at least four-fold more active than imipenem (MIC 90 = 0.5 mg/L) (Table 2; Fig. 1a). A similar pattern was seen against E. coli, Klebsiella spp. and Enterobacter spp. (Table 2). One E. coli isolate (from India) and one Klebsiella oxytoca isolate (from Singapore) was shown to have a high MIC for all three carbapenems (confirmed by the reference laboratory). One Providencia rettgeri isolate (from Thailand) had a doripenem MIC of 4 mg/L determined by the source site but this was not confirmed by the reference laboratory (doripenem MIC 0.12 mg/L at reference laboratory). One E. cloacae isolate (from Thailand) was found to have a doripenem MIC of 1 mg/L (i.e. just above the susceptible breakpoint) by the local laboratory but an MIC < 1 mg/L at the reference laboratory.

The activity of the three carbapenems against Enterobacteriaceae by country is given in Table 3. Doripenem and meropenem were comparable in activity, whilst both were more active than imipenem . Doripenem MICs were ≤0.5 mg/L in isolates from all countries except for the four isolates described above obtained from India, Thailand and Singapore.

A c c e p t e d M a n u s c r i p t 9

Other Gram-negative isolates

Doripenem and meropenem had a lower MIC 50 (MIC for 50% of the organisms) (16 mg/L and 32 mg/L, respectively) than imipenem (≥64 mg/L). All three carbapenems had an MIC 90 of ≥64 mg/L against other Gram-negatives combined (Table 2). None of the carbapenems was effective against Stenotrophomonas maltophilia isolates collected, but 6-8 of 11 isolates of Burkholderia spp. were carbapenem-susceptible (data not shown).

Pseudomonas aeruginosa

Doripenem had the lowest MIC 90 against P. aeruginosa (8 mg/L), with meropenem and imipenem having MICs of ≥64 mg/L (Table 2). Rates of non-susceptibility to doripenem (MIC ≥ 4 mg/L) and meropenem (MIC ≥ 8 mg/L) were the same (21.3%), whilst the rate of non-susceptibility to imipenem (MIC ≥ 8 mg/mL) was 24.6%.

The activity of the three carbapenems against P. aeruginosa by country is given in Table 3.

Pseudomonas aeruginosa isolates from ICU patients tended to have higher carbapenem MICs than those collected outside of the ICU: the MIC 90 for all carbapenems in the ICU was ≥64 mg/L compared with 4 mg/L for doripenem and 16 mg/L for the other two carbapenems outside the ICU (data not shown). Of the P. aeruginosa isolated from patients within the ICU, 30.0% were doripenem nonsusceptible compared with 22.4% of those from outside the ICU (data not shown; see

Carbapenem resistance

Using CLSI breakpoints in place at the time of the study (Table 1) [START_REF]Performance standards for antimicrobial susceptibility testing. Nineteenth informational supplement[END_REF], ca. 17-18% of isolates were non-susceptible to imipenem or meropenem. Currently, the CLSI have not published breakpoints for doripenem, therefore using FDA breakpoints (only susceptible category published), 277 isolates (17.2%) were non-susceptible to doripenem (see Table 1 for breakpoints).

Almost full susceptibility was seen against the Enterobacteriaceae, with nonsusceptibility rates <1% for all carbapenems (data not shown).

All S. maltophilia (n = 8) were non-susceptible to all three carbapenems (likely due to production of metallo--lactamase) and a high percentage of non-susceptibility was seen in Burkholderia spp., Acinetobacter spp. and Pseudomonas spp.

Eighty-six isolates (62.8%) of Acinetobacter spp. were resistant (MIC ≥ 16 mg/L) to both imipenem and meropenem and had high doripenem MICs (4 mg/L to ≥16 mg/L).

One hundred and twenty-four P. aeruginosa isolates (15.9%) were resistant (MIC ≥ 16 mg/L) to both imipenem and meropenem and all but one of these isolates were also doripenem non-susceptible.

Discussion

The in vitro activity of the three carbapenems was determined in this study using Etest methodology. The Etest and BMD MIC values generated by the reference laboratory were in high agreement, and the Etest MIC values generated by each collecting centre were in high agreement with those of the reference laboratory. Etest methodology therefore appears to be a reliable method for doripenem MIC determination.

The in vitro activity of doripenem against Enterobacteriaceae was essentially no different to that of meropenem and was at least four-fold more potent than that of imipenem, including against the important pathogens E. coli and K. pneumoniae. A high percentage of these species are ESBL-producers in Asia-Pacific countries [START_REF] Bell | Doripenem activity against leading bacterial pathogens in the Asia-Pacific region: report from the SENTRY Antimicrobial Surveillance Program[END_REF] and bacteraemic patients with ESBL-positive strains have been shown to have significantly higher average hospital costs, longer median hospital stays following onset of bacteraemia (11 days vs. 5 days; P < 0.001) and higher in-hospital mortality (35% vs. 18%; P = 0.01) compared with patients infected with non-ESBL-producing strains [START_REF] Schwaber | Clinical and economic impact of bacteremia with extended-spectrum--lactamaseproducing Enterobacteriaceae[END_REF]. Doripenem had the lowest MIC distribution of the three carbapenems against P. aeruginosa regardless of whether the isolates were from patients within or outside the ICU and regardless of the type of infection. It is important to note, however, that there was a high proportion of isolates in the collection from Thailand, which may well affect the results of this study. Comparing Thailand with the other countries represented in this study would overestimate resistance in the region because Thailand has higher than average carbapenem MICs. at the time of study [START_REF]Performance standards for antimicrobial susceptibility testing. Nineteenth informational supplement[END_REF], which have not changed in the recent standards update [START_REF]Performance standards for antimicrobial susceptibility testing. Twentieth informational supplement[END_REF].

The exception to this was doripenem, as there are no doripenem breakpoints in the CLSI standards. Organisations that establish breakpoints base their breakpoint decisions on the evaluation of MIC population distributions of organisms to include knowledge of resistance mechanisms and wild-type distributions, pharmacokinetics/pharmacodynamics, and correlation of MIC with clinical outcome.

The organisations evaluate similar types of data but, owing to varying emphasis on key data sets, they may come to different conclusions regarding specific breakpoints.

For example, for doripenem the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints for P. aeruginosa and Enterobacteriaceae are susceptible ≤1 mg/L, intermediate 2-4 mg/L and resistant ≥8 mg/L, whilst the FDA breakpoints are susceptible ≤2 mg/L for P. aeruginosa and ≤0.5 mg/L for Enterobacteriaceae, with no intermediate/resistant categories defined. Other regulatory authorities around the world may also differ in how they establish breakpoints. The choice of the FDA breakpoint for doripenem in this study is a conservative approach as it is two-fold lower than those for meropenem and imipenem for P. aeruginosa and eight-fold lower for Enterobacteriaceae. It is pertinent to note that very recently the CLSI have published an update of the performance standards for antimicrobial susceptibility testing [START_REF]Performance standards for antimicrobial susceptibility testing; update[END_REF], where new carbapenem breakpoints for Enterobacteriaceae are presented, with reduced susceptible, intermediate and resistant breakpoints of ≤1, 2 and ≥4 mg/L, respectively, for all three carbapenems utilised in this study. Re-analysis of the data A c c e p t e d M a n u s c r i p t 13 presented here for the Enterobacteriaceae has little effect on doripenem or meropenem non-susceptibility (0.3-0.6%), but imipenem non-susceptibility increases from 0.2% to 2% (data not shown). At present, no changes to P. aeruginosa CLSI breakpoints have been made, but if a similar change were introduced the relative shift in susceptibility would be in favour of doripenem.

Overall, non-susceptibility to meropenem and imipenem or doripenem was ca. 17-18% with little or no resistance observed in the Enterobacteriaceae. Nine percent of imipenem-resistant and 1% of meropenem-resistant isolates were doripenemsusceptible.

There was greater differentiation between the carbapenems against P. aeruginosa than with other bacterial species. Imipenem resistance in P. aeruginosa varied from country to country. The lowest rate was in Indonesia (5.1%) but this increased to as high as 35-38% in Thailand and India. As the rate of imipenem resistance increased, so did the resistance rate observed for meropenem and doripenem.

Pseudomonas aeruginosa is one of the leading causes of hospital-acquired infections for which treatment options are limited [START_REF] Thoracic | Guidelines for the management of adults with hospital-acquired, ventilatorassociated, and healthcare-associated pneumonia[END_REF], and MDR P. aeruginosa has a significant impact on mortality and costs [START_REF] Harris | Epidemiology and clinical outcomes of patient with multiresistant Pseudomonas aeruginosa[END_REF][START_REF] Tacconelli | Multidrug-resistant Pseudomonas aeruginosa bloodstream infections: analysis of trends in prevalence and epidemiology[END_REF][START_REF] Carmeli | Health and economic outcomes of antibiotic resistance in Pseudomonas aeruginosa[END_REF]. In addition, an increased carbapenem (imipenem and meropenem) MIC will clearly reduce the therapeutic options for P. aeruginosa infections where high levels of antibiotic resistance are common [START_REF] Bell | Doripenem activity against leading bacterial pathogens in the Asia-Pacific region: report from the SENTRY Antimicrobial Surveillance Program[END_REF][START_REF] Tacconelli | Multidrug-resistant Pseudomonas aeruginosa bloodstream infections: analysis of trends in prevalence and epidemiology[END_REF]. It has been postulated that extending the infusion time of a more active agent may overcome these difficulties by increasing the likelihood that the pharmacodynamic target is attained [START_REF] Nicolau | Carbapenems: a potent class of antibiotics[END_REF]. Furthermore, in vivo simulations support A c c e p t e d M a n u s c r i p t 14 the use of prolonged infusions of doripenem at 1 g or 2 g doses as a potential treatment for P. aeruginosa isolates with raised doripenem MICs [START_REF] Crandon | In vivo efficacy of 1-and 2-gram human simulated prolonged infusions of doripenem against Pseudomonas aeruginosa[END_REF].

Although the in vitro activity of doripenem against Pseudomonas appears to be the best of the three carbapenems tested, geography and whether the patient is in or outside the ICU was shown to impact this activity. A real-time surveillance programme monitoring the susceptibility and MICs of these bacterial pathogens at the country or healthcare centre level appears to be a necessary tool to guide empirical treatment effectively. The Etest method for MIC determination is a rapid, convenient and reliable method for use in the clinical diagnostic laboratory.

Confirmatory testing using a reference BMD method could be performed as required.

Because in vitro data do not always extrapolate to the clinical setting, prospective controlled clinical trials are needed to demonstrate the efficacy of different infusion regimens or administration. Furthermore, there is generally a high prevalence of antibiotic resistance in Gram-negative bacteria in the Asia-Pacific region that will continue to be a problem for clinicians. These challenges will need to be overcome by alternative strategies with existing agents such as dosing manipulation (e.g. higher doses and/or prolonged infusion with doripenem) or combination therapy.

  of Carbapenems Testing (COMPACT) Study was designed to determine the in vitro potency of doripenem compared with imipenem and meropenem against a large number of contemporary Gram-negative pathogens from more than 100 centres across Europe and the Asia-Pacific region and to assess the reliability of Etest methodology for doripenem minimum inhibitory concentration (MIC)

  Streptococcus pneumoniae), Enterobacteriaceae, Haemophilus influenzae, P. aeruginosa, Moraxella catarrhalis, ceftazidime-susceptible Acinetobacter spp., Bordetella spp., Bacteroides spp., Prevotella spp., Clostridium spp. and other Gram-positive anaerobes, including difficult-to-treat pathogens [14]. Doripenem is approved for the treatment of complicated urinary tract infections and complicated intra-abdominal infections (cIAIs) in the USA, Europe and Asia-Pacific region, and for nosocomial pneumonia (NP) [including ventilator-associated pneumonia (VAP)] both in Europe and several Asia-Pacific countries such as Thailand, Philippines, Malaysia, Australia and Indonesia.

A c c e p t e d M a n u s c r i p t 7 baumannii

 7 (74.4%) was the most isolated species amongst the other Gram-negatives collected.

  A c c e p t e d M a n u s c r i p t 8 cumulative percentage inhibition curves for Enterobacteriaceae, A. baumannii and P. aeruginosa.

A c c e p t e d M a n u s c r i p t 10 The

 10 carbapenems were equally active against P. aeruginosa isolates from BSIs, cIAIs and NP (data not shown).

A c c e p t e d M a n u s c r i p t 12 Susceptibility

 12 analyses in this study were evaluated using breakpoints developed by the internationally recognised performance standards developing organisation (CLSI)

20 Fig. 1 .

 201 Fig. 1. Cumulative minimum inhibitory concentration (MIC) distribution for doripenem,

Table 2

 2 summarises the in vitro activity of doripenem, imipenem and meropenem by Etest methodology as reported by the collecting centres, and Fig.1provides the

Table 1 for

 1

breakpoints).

Table 1

 1 Breakpoints (in mg/L) used to determine susceptibility of isolates for minimum

	inhibitory concentration testing
	Isolates	FDA	CLSI
		Doripenem Imipenem	Meropenem
		S	I R S I R	S	I R
	Pseudomonas aeruginosa	≤2	--≤4 8 ≥16 ≤4	8 ≥16
	Enterobacteriaceae	≤0.5 --≤4 8 ≥16 ≤4	8 ≥16
	Acinetobacter spp. (A. baumannii) ≤1	--≤4 8 ≥16 ≤4	8 ≥16
	Stenotrophomonas maltophilia	≤2	--≤4 8 ≥16 ≤4	8 ≥16
	Burkholderia cepacia	≤2	--≤4 8 ≥16 ≤4	8 ≥16
	Other non-Enterobacteriaceae	≤1	--≤4 8 ≥ 16 ≤4	8 ≥16
	Haemophilus influenzae	≤1	--≤4 --	≤0.5 --
	FDA, US Food and Drug Administration; CLSI, Clinical and Laboratory Standards
	Institute; S, susceptible, I, intermediate; R, resistant.

Table 2

 2 Summary minimum inhibitory concentration (MIC) data for doripenem (DOR), One K. oxytoca isolate was less susceptible to all three carbapenems (confirmed by the reference laboratory), with DOR, IPM and MEM MIC values 4, 2 and 8 mg/L; the highest MICs excluding this isolate were 0.5, 1 and 0.5 mg/L, respectively.

	Edited Table 2
	imipenem (IPM) and meropenem (MEM) against Gram-negative bacteria as
	determined using Etest methodology by the collecting centres
	Isolates (n)	Antimicrobial	MIC (mg/L)
		agent	MIC 50 MIC 90 Range
	Enterobacteriaceae (656)	DOR	0.03	0.06	0.008-8 a,b
		IPM	0.25	0.5	0.008-8 a,b
		MEM	0.03	0.12	0.008-16 a,b
	Escherichia coli (253)	DOR	0.015 0.06	0.008-8 a
		IPM	0.12	0.25	0.015-8 a
		MEM	0.03	0.06	0.008-16 a
	Klebsiella spp. (243)	DOR	0.03	0.06	0.008-4 b
		IPM	0.25	0.25	0.008-2 b
		MEM	0.03	0.06	0.008-8 b
	Enterobacter spp. (68)	DOR	0.03	0.25	0.015-1
		IPM	0.25	1	0.015-4
		MEM	0.03	0.25	0.015-2
	Salmonella spp. (30)	DOR	0.015 0.03	0.008-0.25
		IPM	0.12	0.25	0.03-1
		MEM	0.015 0.03	0.008-0.03
	Proteus spp. (21)	DOR	0.06	0.25	0.03-0.25
		IPM	0.5	2	0.25-4
		MEM	0.06	0.25	0.03-0.5
	Serratia spp. (13)	DOR	-	-	0.015-0.12
		IPM	-	-	0.25-1
		MEM	-	-	0.03-0.12
	Citrobacter spp. (10)	DOR	-	-	0.015-0.5
		IPM	-	-	0.12-2
		MEM	-	-	0.015-0.5
	Pseudomonas aeruginosa (780)	DOR	0.25	8	0.002 to
					≥64

a One E. coli isolate was less susceptible to all three carbapenems (confirmed by the reference laboratory), with DOR, IPM and MEM MIC values of 8, 8 and 16 mg/L; the highest MICs excluding this isolate were 0.5, 1 and 0.5 mg/L, respectively. b

Table 3

 3 Carbapenem minimum inhibitory concentrations (MICs) (mg/L) against Pseudomonas aeruginosa and Enterobacteriaceae isolates evaluated by country

	Edited Table 3
	Country	Carbapenem P. aeruginosa (n = 780)	Enterobacteriaceae (n = 656)
			n	MIC 50 MIC 90 Range	n	MIC 50 MIC 90 Range
	Australia	DOR	87 0.25	4	0.03 to	82 0.015 0.03	0.008-
						≥64			0.12
		IPM		1	8	0.06 to		0.25	0.25	0.12-2
						≥64
		MEM		0.5	8	0.015 to		0.03	0.03	0.008-
						≥64			0.25
	Hong	DOR	59 0.25	2	0.06-8	47 0.03	0.12	0.015-
	Kong								0.5
		IPM		1	2	0.5 to		0.25	1	0.03-4
						≥64
		MEM		0.25	2	0.03-16		0.03	0.12	0.015-
									0.25
	India	DOR	66 0.25	≥64	0.03 to	65 0.015 0.06	0.008-8
						≥64
		IPM		2	≥64	0.12 to		0.12	0.25	0.06-8
						≥64
		MEM		0.5	≥64	0.06 to		0.03	0.06	0.008-
						≥64			16
	Indonesia DOR	39 0.25	4	0.03 to	36 0.06	0.25	0.008 -
						≥64			0.5
		IPM		2	4	0.12 to		0.25	1	0.03-4
						≥64
		MEM		0.5	4	0.03 to		0.06	0.5	0.008-1
						≥64
	Malaysia	DOR	89 0.25	2	0.015 to	71 0.03	0.06	0.008-
						≥64			0.5

Acknowledgments

The authors would like to thank Lucie Farmer and Sarah Farrell (Quotient Bioresearch Ltd., Fordham, UK) for laboratory support as well as Mike Stek for scientific and strategic support. They also thank the many investigators in each country who participated in the collection of isolates (COMPACT Asia-Pacific Study Group): J. Turnidge and J. Bell (Women's and Children's Hospital, Adelaide,

Funding

The research outlined in this publication was funded by Janssen-Cilag.

Competing interests

Quotient Bioresearch Ltd. (Fordham, UK) carries out research on behalf of numerous pharmaceutical companies for the development of new antimicrobials.

Ethical approval