Detection of β-alanyl aminopeptidase as a biomarker for: Pseudomonas aeruginosa in the sputum of patients with cystic fibrosis using exogenous volatile organic compound evolution

Article abstract of DOI:10.1039/c9ra08386c

A novel, rapid and sensitive analytical method has been developed and applied to 105 sputum samples from patients with cystic fibrosis, including 5 samples from post-lung transplant patients. This new method is specifically targeted to measure β-alanyl am

Full text of DOI:10.1039/c9ra08386c

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Detection of b-alanyl aminopeptidase as
a biomarker for Pseudomonas aeruginosa in the
sputum of patients with cystic fibrosis using
exogenous volatile organic compound evolution
Cite this: RSC Adv., 2020, 10, 10634
b
Ryan Thompson,a Dominic Stephenson,ab Hannah E. Sykes,a John D. Perry,
a
Stephen P. Stanfortha and John R. Dean
*
A novel, rapid and sensitive analytical method has been developed and applied to 105 sputum samples from
patients with cystic fibrosis, including 5 samples from post-lung transplant patients. This new method is
specifically targeted to measure b-alanyl aminopeptidase activity which is characteristic of some
important Gram-negative pathogens. Of relevance to this study are Pseudomonas aeruginosa and
pathogens of the Burkholderia cepacia complex both of which are commonly associated with
respiratory infections as well as increased morbidity and mortality in adult cystic fibrosis patients. The
analytical method involves the addition of a novel enzyme substrate (i.e. 3-amino-N-(3-fluorophenyl)
propanamide) that interacts with b-alanyl aminopeptidase to generate an exogenous volatile organic
compound 3-fluoroaniline (LOD 0.02 mg mLꢀ1; LOQ 0.06 mg mLꢀ1). 3-Fluoroaniline was determined at
20 times above its calculated limit of quantification in the sputum samples by HS-SPME-GC-MS and
then the results compared with standard culture methods and bacterial identification using MALDI-TOF-
MS. Detection of 3-fluoroaniline was possible after only 8 h incubation of the sputum samples with
a 95% success rate; this increased to 100% at 24 h which was well within the typical routine timeframe
of 48 h. To our knowledge, this is the first demonstration of detection of P. aeruginosa by use of
a custom-designed substrate to liberate a detectable and unique VOC. The very high negative predictive
value (100% in this study) means such an assay could be appropriate as a screening technique for
patients who are not yet colonized by this pathogen.
Received 14th October 2019
Accepted 2nd March 2020
DOI: 10.1039/c9ra08386c
rsc.li/rsc-advances
airways and are very susceptible to further lung colonization
and bacterial infections. In older children the rst symptoms
Introduction
are oen a mix of abnormal liver function test results, pancre-
atic exocrine insufficiency, an absence of vas deferens devel-
opment in males, and persistent Staphylococcus aureus
respiratory infections. As patients age into young adulthood,
their symptoms oen progress into cirrhosis of the liver due to
chronic inammation, intermittent respiratory infections from
Pseudomonas aeruginosa, and established bronchiectasis. From
approximately age 20 onwards, patients will frequently have
established bronchiectasis associated with persistent respira-
tory infections with Gram-negative bacteria such as Pseudo-
monas aeruginosa and strains of the Burkholderia cepacia
complex. The necessity for lung transplantation is frequently
required for patients aged 30 onwards.4
Prognosis for CF patients is relatively poor in comparison to
healthy individuals, although is improving along with better
medical intervention. Based on data from the UK Cystic Fibrosis
Registry, half of CF patients born in 2017 would have a life
expectancy of approximately 47 years based upon current
treatment and management regimes. However, in 2017 the
Cystic brosis (CF) was rst identied as a distinct condition in
1938 by Dr Dorothy Anderson, who observed a pattern of cystic
brosis of the pancreas in patients with a history of respiratory
issues, subsequently leading to the identication of cystic
brosis as a distinct condition.1 CF is a common autosomal
recessive congenital disorder caused by mutations in the cystic
brosis transmembrane conductance regulator (CFTR) gene
responsible for anion transport, predominantly chloride &
bicarbonate, across epithelial cell membranes and mucociliary
clearance in respiratory airways.2
The absence or reduced function of the CFTR gene results in
extra thick mucosal secretions in organ systems with epithelial
cell linings, such as the lungs, digestive system, hepatobiliary
tracts, and vas deferens in males.3 Due to this abnormally
viscous lining, CF patients struggle to clear mucus from their
aDepartment of Applied Sciences, Northumbria University, Ellison Building, Newcastle
upon Tyne, NE1 8ST, UK. E-mail: John.Dean@northumbria.ac.uk
bDepartment of Microbiology, Freeman Hospital, Newcastle upon Tyne, NE7 7DN, UK
10634 | RSC Adv., 2020, 10, 10634–10645
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median age of death for CF patients in the UK was reported as Canada, B. multivorans is the most prevalent.20 Interestingly,
31 years.5 The most common cause of death in CF patients is Burkholderia gladioli, a non B. cepacia complex strain member of
respiratory failure secondary to progressive lung disease, oen the Burkholderia genus, has become more prominent in US CF
caused by chronic infection. Due to an innate inability to clear patients, but is very uncommon elsewhere.20
the thick mucus associated with the condition, CF patients are
Pseudomonas and Burkholderia infections are predominantly
susceptible to colonization by opportunistic pathogens that diagnosed in CF patients through the culturing of patient
pose little risk to non-compromised individuals. The pathology respiratory samples, such as sputum, cough swab, and bron-
associated with failure to clear microorganisms is exacerbated choalveolar lavages. Detection via culture relies on a plethora of
by the patient's immune response, causing chronic inamma- different selective media, as there is no one denitive protocol
tion and tissue damage.6 In younger patients, the primary for detection in hospitals so selective media panels and proto-
bacteria responsible for infection are Staphylococcus aureus and cols are oen developed in-house. Whilst culture methods have
Haemophilus inuenzae,7 both of which cause signicant proved successful thus far for the isolation of Pseudomonas and
damage to the airways either directly, or indirectly through the Burkholderia, results are oen only available following 48–72 h
immune inammatory response.8 As the condition progresses, incubation, which when combined with the importance of
patients develop chronic bronchiectasis and become more beginning patient treatment early, could be detrimental to
susceptible to infection from Gram-negative bacteria commonly patient prognosis. The chromogenic culture medium
found in the environment, such as Achromobacter sp., Steno- CHROMID® Pseudomonas contains a chromogenic substrate for
trophomonas maltophilia, Pseudomonas aeruginosa, and Bur- b-alanyl aminopeptidase, which is hydrolysed by P. aeruginosa
kholderia cepacia complex strains.9,10 Additionally, there is an resulting in the formation of purple colonies aer 24 to 48 h of
increase in prevalence of nontuberculous Mycobacterium infec- incubation.22 For specimens from cystic brosis patients,
tions in patients with cystic brosis, particularly Mycobacterium incubation can be extended up to 5 days. Our aim was to
abscessus and Mycobacterium avium-intracellulare.11,12
therefore develop a rapid test for the detection of Pseudomonas
Pseudomonas aeruginosa is by far the most common path- and Burkholderia from CF patient sputum samples, utilising b-
ogen associated with respiratory infections in CF patients. In alanyl aminopeptidase activity. We have previously identied
2015, the UK national cystic brosis registry reported 49% of CF that using targeted enzyme substrates, a unique exogenous
patients aged 18 and above had a chronic P. aeruginosa infec- volatile organic compound can be liberated and detected using
tion,13 however some studies report the incidence of Pseudo- either static headspace-solid phase micro-extraction-gas
monas infection as high as 60% in adult patients.14 Some studies chromatography-mass spectrometry, SHS-SPME-GC-MS23–26 or
have shown that infection with P. aeruginosa can occur very early static headspace multicapillary column-gas chromatography-
in childhood. In longitudinal studies on children diagnosed ion mobility spectrometry, SHS-MCC-GC-IMS.27 The use of
with CF, annual bronchoalveolar lavage cultures have shown exogenous volatile organic compounds (VOCs) to distinguish 7
a high prevalence of P. aeruginosa (ꢁ11%).7,15 However, a further Gram-positive from 15 Gram-negative bacteria using the
study which monitored the presence of P. aeruginosa antibodies enzyme substrates 2-amino-N-phenylpropanamide (to liberate
found a higher incidence of infection at a younger age, as well as the VOC aniline) and 2-amino-N-(4-methylphenyl)propanamide
in patients who tested culture negative suggesting that some P. (to liberate the VOC p-toluidine) has been demonstrated.24
aeruginosa infections may be cleared naturally in younger Similarly the approach has been applied for the analysis of
patients.16 The early detection and treatment of P. aeruginosa enzyme activity in Listeria species using the enzyme substrates
infection is crucial and highly associated with a good prognosis. benzyl-a-D-mannopyranoside (to liberate the VOC benzyl
Early intervention with a strict antibiotic regime has been alcohol) and D-alanyl 3-uoroanilide (to liberate the VOC 3-u-
shown to achieve eradication of P. aeruginosa infection in oroaniline).25 In addition, exogenous VOC detection has been
approximately 60% of cases,17 however if this short window is used to identify Salmonella in milk samples using the enzyme
missed, then infections are considered virtually impossible to substrates 2-chlorophenyl octanoate, phenyl a-D-galactopyr-
eliminate.17 Chronic P. aeruginosa infections have clear associ- anoside and L-pyrrolidonyl uoroanilide which liberate the
ations with an accelerated decline in lung function,18 and VOCs 2-chlorophenol, phenol and 3-uoroaniline, respec-
increased mortality.19
tively.26 In this case however, b-alanyl aminopeptidase enzymes
Another important pathogen in CF patients are members of work by catalysing the cleavage, via hydrolysis, of amino acids
the Burkholderia cepacia complex, a group of 22 members of the from the amino (N) terminus of protein or peptide substrates.28
Burkholderia genus. Infections from B. cepacia strains are The presence of b-alanyl aminopeptidase activity is well docu-
strongly associated with a severe decline in lung function in CF mented in P. aeruginosa,29,30 and has subsequently led to studies
patients, and an increased mortality rate.20 Patients infected on the development of chromogenic and uorogenic substrates
with B. cepacia complex strains may also develop a life threat- targeting b-alanyl aminopeptidase activity as a method for the
ening systemic infection known as cepacia syndrome, where detection of P. aeruginosa.31,32 There is also further evidence for
eradication of the infection is impossible and has been highly b-alanyl aminopeptidase activity in Burkholderia cepacia
associated with post lung transplant mortality.21 The most complex and Serratia marcescens in studies using the b-alanyl
common members of the B. cepacia complex strains associated based 7-N-(b-alanyl)aminophenoxazin-3-one,31 9-(40-N-[b-alanyl]
with CF patients across Europe and Australasia are B. multi- aminophenyl)acridines,33 and 9-(40-N-[b-alanyl]aminophenyl)-
vorans and B. cenocepacia, whereas in the United States and 10-methylacridinium salts.34
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reference database version 3.1.2.0. Isolated colonies were
identied down to either genus or species level through the
comparison of the sample generated spectra against that of an
in-house taxonomy spectra library. All distinct colony
morphologies isolated from the agar plates were subject to
analysis via MALDI-TOF-MS by initially transferring a thin lm
of the colony onto a single stainless steel target plate position.
The transferred colonies were covered with 1 mL of 70% formic
acid then once dried, 1 mL HCCA matrix solution: alpha-cyano-
4-hydroxycinnamic acid (10 mg HCCA dissolved in a solution of
475 mL distilled water, 500 mL acetonitrile, and 25 mL triuoro-
acetic acid). Following the addition of the matrix solution, the
samples were air dried prior to MALDI-TOF-MS analysis.
Experimental
Instrumentation and analysis
Gas chromatography-mass spectrometry (GC-MS) analyses were
performed using a ThermoFinnigan Trace GC Ultra paired with
a Polaris Q ion trap mass spectrometer (Thermo Fisher scien-
tic, Loughborough, UK) with Xcalibur 1.4 SR1 soware
package (Thermo Fisher). Separation of the analytes was ach-
ieved using an Agilent Technologies (Wokingham, UK) DB-5MS
column (30 m ꢂ 0.25 mm internal diameter ꢂ 0.25 mm lm
thickness), with the following temperature program: initial
oven temperature 50 ꢃC (hold 2 min), then a ramp to 250 ꢃC @
12.5 ꢃC minꢀ1, followed by a nal hold time of 2 min. The
Polaris Q ion trap mass spectrometer was set to full scan mode,
scanning a mass range of 33–200 m/z, with a scan event time of
Materials & reagents
ꢃ
0.31 s. The ion source temperature was maintained at 260 C,
and the mass transfer line was maintained at 250 C. Identi-
Brain heart infusion broth (CM1136), brain heart infusion agar
(CM1135), Columbia blood agar base (CM0331), CLED medium
with Andrades indicator (CM0423), Sabouraud dextrose agar
(CM0041), bacteriological agar no. 1 (LP001B), and Sputasol
(SR0233A) were all purchased from Oxoid, Basingstoke, UK.
Bacitracin (11072), chloramphenicol (C0378), Middlebrook 7H9
broth base (M0178), glycerol (G5516), 85 mm polyacrylate sol-
idphase micro-extraction bres, and N-methyl pyrrolidine
(NMP) (328634) were purchased from Sigma, Gillingham, UK.
Defribrinated horse blood (5% v/v, HB035) was purchased from
TCS biosciences, Buckingham, UK. Burkholderia cepacia selec-
tive agar (PP0160) was purchased from EO laboratories, Bon-
nybridge, UK. Yeast extract (03904110) was purchased from
ꢃ
cation of VOCs was achieved using the National Institute of
Standards and Technology (NIST) reference library (NIST Mass
spectral library, version 2.0a, 2001) as well as the comparison of
the retention times and mass spectra of authentic standards. All
samples and standards were maintained at 37 ꢃC during SPME
sampling through the use of a water bath. All sputum samples
were stored within a 37 ꢃC incubator prior to analysis, and were
placed in the water bath 10 min prior to SPME analysis using an
85 mm polyacrylate bre (Sigma-Aldrich, Poole, UK) for
temperature maintenance. Standards which were not previously
stored at 37 ꢃC were placed in the water bath for a minimum of
30 min prior to sampling. Fibres were used for analyte extrac-
tion with a manual holder, and were exposed in the headspace
above all samples and standards for a total of 10 min. Following
the adsorption of analytes, the bre was immediately retracted
inside the needle and transported directly to the inlet of the GC-
MS. Analyte desorption was carried out by exposing the laden
bre within the split-splitless GC injection port at 250 ꢃC for
2 min. The inlet was set to split mode with a split ratio of 1 : 10,
with the helium carrier gas ow rate set to 1 mL minꢀ1. Fibres
were used for a maximum of 125 injections to ensure consis-
tency of readings. The same SPME bre type, method, and GC-
MS program was used for all samples, blanks, and standards,
throughout this study. The limit of detection (LOD) and limit of
quantication (LOQ) for HS-SPME-GC-MS were determined by
calculating the standard deviation (n ¼ 7) of the background
noise from the same retention time as the analyte. The LOD was
determined by multiplying the standard deviation by 3, and the
LOQ determined by multiplying by 10. Calibration curves were
determined by running known concentrations of each exoge-
nous VOC standard ranging from 0–100 mg mLꢀ1, giving a y ¼
mx + c value for sample concentration calculations. Due to
variability of adsorptive efficiency within the bres, a new cali-
bration for each analyte was required each time a new bre was
used.
´
´
bioMerieux, Marcy-l'Etoile, France. All bacteria used in this
study were acquired from the Freeman Hospital Microbiology
Department, Newcastle upon Tyne, UK. All P. aeruginosa strains
(except NCTC 12903) were all strains isolated from patients with
cystic brosis globally. All Burkholderia strains were part of the
Burkholderia cepacia complex.
Procedures for routine microbiological testing of cystic
brosis patient samples
Patient sputum specimens are routinely received for culture by
the Freeman Hospital Microbiology Department from clinics
which are run for CF patients. The samples used in this study
were aliquots of leover samples referred to the Microbiology
Department for routine analyses. No additional samples were
taken for the purposes of this study. Samples were anonymized,
except for the gender and age of the patient, by laboratory staff
with legitimate access to patient data. Anonymization meant
that patients could not be identied, and the results of this
independent study could not be used to affect patient
management in any way. Strict adherence to these conditions
meant that patient consent was not sought, in accordance with
the Medical Research Council Operational and Ethical Guide-
lines on the use of Human Tissue and Biological Samples for
Use in Research.
Matrix Assisted Laser Desorption Ionization-Time of Flight-
Mass Spectrometry (MALDI-TOF-MS) analysis was performed
by a Microex LT mass spectrometer (Bruker Daltonik) using
the MALDI Biotyper soware package (version 3.0) with the
Upon sample reception, the sputum specimens were mixed
with an equal volume of sterile Sputasol and vortexed thor-
oughly until homogenization occurred. Homogenized samples
were cultured onto 8 different agar media to ensure good
10636 | RSC Adv., 2020, 10, 10634–10645
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differentiation and identication of organisms potentially horse blood (TCS biosciences; HB035, Buckingham, UK) was
present. Then, 1 mL aliquots of the homogenous specimens added. Filter sterilised aztreonam (Azactam) was added to the
were transferred into sterile 20 mL glass vials and refrigerated at mixture at 15.625 mg Lꢀ1 before dispensing. Plates were
ꢃ
4 C until sample collection days.
streaked using a 10 mL loop. Plates were incubated within
Blood agar. Columbia blood agar base (Oxoid; CM0331, a non-modied air atmosphere, and maintained at 37 ꢃC.
Basingstoke, UK) was mixed with distilled water to a concen- Results were visually interpreted at both 24 and 48 h
tration of 39 g Lꢀ1 and autoclaved at 121 ꢃC for 15 min, incubation.
following sterilisation 5% (v/v) debrinated horse blood (TCS
Burkholderia cepacia agar (CEP). Burkholderia cepacia
biosciences; HB035, Buckingham, UK) was added before selective agar was purchased ready-made (E&O labs, PP0160,
dispensing approximately 20 mL per plate. Plates were streaked Bonnybridge, UK). Plates were inoculated through the lling of
using a 10 mL loop. Plates were then incubated within a constant a 3 mL pastette with the sputum sample, and subsequently
ꢃ
5% CO2 atmosphere, and maintained at 37 C.
streaking 2 drops. Plates were incubated within a non-modied
Chocolate agar with bacitracin (CBAC). Columbia blood agar air atmosphere, and maintained at 37 ꢃC. Results were visually
base (Oxoid; CM0331, Basingstoke, UK) was mixed with distilled interpreted following 5 days of incubation.
water to a concentration of 39 g Lꢀ1 and autoclaved. Following
Rapidly growing mycobacterium agar (RGM). RGM agar was
sterilisation, 5% (v/v) debrinated horse blood (TCS biosci- prepared as previously described.35
ences; HB035, Buckingham, UK) was added, and the mixture
was reheated to 80 ꢃC to achieve haemolysis. Filter sterilised
Individual substrate testing against pure Pseudomonas
aeruginosa and Burkholderia cepacia complex strains
bacitracin (Sigma; 11702) dissolved in N-methyl 2-pyrrolidone
(NMP) was then added to a nal concentration of 70 mg Lꢀ1
,
and the mixture dispensed to approximately 20 mL per plate. Sample vials were prepared by rst suspending bacterial cells in
Plates were streaked using a 10 mL loop. Plates were incubated sterile Brain Heart Infusion (BHI) broth and adjusting the
within a constant 5% CO2 atmosphere, and maintained at absorbance to 0.132 when measured at 600 nm. This gave an
37 ꢃC. Results were visually interpreted at both 24 and 48 h inoculum of approximately 1–1.5 ꢂ 108 CFU mLꢀ1, equivalent
incubation.
to 0.5 McFarland units. The sterile BHI broth was prepared by
Chocolate agar. Columbia blood agar base (Oxoid; CM0331, dissolving 37 g of the powder mix into 1 litre of MilliQ water,
Basingstoke, UK) was mixed with distilled water to a concen- and sterilising the resulting mix via autoclave at 121 ꢃC for
tration of 39 g Lꢀ1 and autoclaved. Following sterilisation, 5% 15 min. Following this, 100 mL of 1 ꢂ 108 CFU mLꢀ1 stock was
(v/v) debrinated horse blood (TCS biosciences; HB035, Buck- added to 9.89 mL of sterile BHI broth. To this new bacterial
ingham, UK) was added, and the mixture was reheated to 80 ꢃC suspension, 10 mL of the chosen substrate i.e. 3-amino-N-(3-
to achieve haemolysis. The mixture was then dispensed to uorophenyl)propanamide, 3-amino-N-(4-methylphenyl)prop-
approximately 20 mL per plate. Plates were inoculated by rst anamide or 3-amino-N-phenylpropanamide (dissolved in NMP
inoculating 2 mL of sterile water with 1 mL loop of sputum to 100 mg mLꢀ1) was added. The nal volume of the sample
sample, from this inoculated water a 10 mL loop was used for vials was 10 mL, with a nal bacterial inoculum of 1 ꢂ 106 CFU
streaking. Plates were incubated within a constant 5% CO2 mLꢀ1, and a nal substrate concentration of 100 mg mLꢀ1
.
atmosphere, and maintained at 37 ꢃC. Results were visually Sterile 20 mL glass vials with septum lids were used for all
interpreted at both 24 and 48 h incubation. samples. Once prepared, the sample vials were placed into an
Cystine–lactose–electrolyte decient agar (CLED). CLED incubator set to 37 ꢃC overnight, ranging from 20–24 h.
medium with Andrades indicator (Oxoid; CM0423, Basingstoke, Following overnight incubation, the samples were then ana-
UK) was mixed with distilled water to 36.2 g Lꢀ1 and autoclaved. lysed via HS-SPME-GC-MS.
Following sterilisation, the mixture was dispensed as standard.
Plates were streaked using a 10 mL loop. Plates were incubated
Procedure for analysis of cystic brosis patient sputum
samples by HS-SPME-GC-MS
within a non-modied air atmosphere and maintained at 37 ꢃC.
Results were visually interpreted at both 24 and 48 h incubation.
Sabouraud agar with chloramphenicol (SAB). Sabouraud On sample collection days, 10 mL of BHI broth containing 100
dextrose agar (Oxoid; CM0041, Basingstoke, UK) was mixed with mg mLꢀ1 of 3-amino-N-(3-uorophenyl)propenamide, 5 mg Lꢀ1
distilled water to 65 g Lꢀ1 and autoclaved. Following auto- amphotericin B (an anti-fungal) and 70 mg Lꢀ1 bacitracin (a
claving, lter sterilised chloramphenicol heated at 100 ꢃC for combination of peptides that inhibit the growth of yeasts and
5 min (Sigma; C0378) was added to the mixture at 50 mg Lꢀ1
,
Gram-positive bacteria) was added to sample vials containing
and the mixture dispensed. Plates were streaked using a 10 mL 1 mL specimen and then vortexed to thoroughly mix. Samples
loop. Plates were incubated within a non-modied air atmo- were then collected and transported to the University Labora-
ꢃ
sphere and maintained at 37 ꢃC. Results were visually inter- tories (within 1 h) where they were incubated at 37 C for set
preted following 5 days of incubation.
time periods before analysis. Great care was taken to not disturb
Aztreonam selective agar (AZTRE). Columbia blood agar or move the lids of the vials. Samples were placed into the
base (Oxoid; CM0331, Basingstoke, UK) was mixed with incubator in 20 min intervals, to account for the GC-MS runtime
distilled water to a concentration of 39 g Lꢀ1 and autoclaved at and ensure that all samples were analysed following either 8 h
121 ꢃC for 15 min, following sterilisation 5% (v/v) debrinated or 24 h incubation.
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with diethyl ether giving the desired products (as their TFA
salts) as white, hydroscopic solids.
Procedures for synthesis of enzyme substrates
The b-alanyl substrates 1a–c [as their triuoroacetate (TFA)
salts] were prepared from their corresponding Boc-protected
derivatives 2a–c.
Analytical data
3-Amino-N-(4-methylphenyl)propanamide, TFA salt 1b. 1H-
NMR (400 MHz; DMSO-d6) d: 10.1 (1H, s, N–H), 7.93 (3H,
+
broad s, –NH3 ), 7.44 (2H, d, J ¼ 8.8 Hz, Ar–H), 7.06 (2H, d, J ¼
8.8 Hz, Ar–H), 3.04 (2H, m, CH2), 2.66 (3H, t, J ¼ 6.8 Hz, CH2),
2.2 (3H, s, CH3); 13C-NMR (101 MHz; DMSO-d6) d: 168.6 (C]O),
159.1 (q, J ¼ 31 Hz, TFA), 136.9 (Ar–C), 132.7 (Ar–C), 129.6 (2 ꢂ
Ar–C), 119.9 (2 ꢂ Ar–C), 118.9 (q, J ¼ 297 Hz, TFA), 35.5 (CH2),
33.7 (CH2), 20.9 (CH3); HRMS: m/z found, 179.1177. C10H15N2O
[MH]+ requires, 179.1184.
3-Amino-N-(3-uorophenyl)propanamide, TFA salt 1c. 1H-
NMR (400 MHz; DMSO-d6) d: 10.42 (1H, s, N–H), 7.88 (3H,
broad s, –NH3 ), 7.57 (1H, d, J ¼ 11.6 Hz, Ar–H), 7.31–7.26 (2H,
Compounds 2a–c were synthesised from b-alanyl and an
+
appropriate aniline derivative using
a mixed anhydride
m, Ar–C), 6.84 (1H, t, J ¼ 7.6 Hz, Ar–H), 3.05 (2H, broad s, CH2),
2.68 (2H, t, J ¼ 6.8 Hz, CH2); 13C-NMR (101 MHz; DMSO-d6) d:
169.3 (C]O), 162.6 (d, J ¼ 240 Hz, Ar–C), 141.1 (d, J ¼ 10 Hz, Ar–
C), 130.9 (d, J ¼ 10 Hz, Ar–C),115.4 (Ar–C), 110.3 (d, J ¼ 21 Hz,
Ar–C), 106.5 (d, J ¼ 27 Hz, Ar–C), 35.3 (CH2), 33.8 (CH2); HRMS:
m/z found, 183.0929. C9H12N2OF [MH]+ requires, 183.0934.
tert-Butyl [3-(4-methylanilino)-3-oxopropyl]carbamate 2b.
1H-NMR (400 MHz; DMSO-d6) d: 9.80 (1H, broad s, N–H), 7.42
(2H, d, J ¼ 8.8 Hz, Ar–H), 7.03 (2H, d, J ¼ 8.8 Hz, Ar–H), 6.82 (1H,
broad s, N–H), 3.16 (2H, dt, J ¼ 6.9 and 6.9 Hz, CH2), 2.39 (2H, t,
J ¼ 6.9 Hz, CH2), 2.19 (3H, s, CH3), 1.33 (9H, s, (CH3)3); 13C-NMR
(101 MHz; DMSO-d6) d: 169.6 (C]O), 156.1 (C]O), 137.2 (Ar–
C), 132.4 (Ar–C), 129.5 (2 ꢂ Ar–C), 119.6 (2 ꢂ Ar–C), 78.1 (C-But),
37.2 (CH2), 37.1 (CH2), 28.8 (3 ꢂ CH3), 21.0 (CH3).
coupling reaction. tert-Butyl (3-anilino-3-oxopropyl)carbamate
2a and 3-amino-N-phenylpropanamide (TFA salt) 1a have been
described previously.36
1
tert-Butyl [3-(3-uoroanilino)-3-oxopropyl]carbamate 2c. H-
NMR (400 MHz; DMSO-d6) d: 10.11 (1H, broad s, N–H), 7.57 (1H,
d, J ¼ 12.0 Hz, Ar–H), 7.28–7.25 (2H, m, Ar–H), 6.85–6.80 (2H, m,
Ar–H and N–H), 3.18 (2H, dt, J ¼ 7.2 and 7.2 Hz, CH2), 2.44 (2H,
t, J ¼ 7.2 Hz, CH2), 1.33 (9H, s, (CH3)3); 13C-NMR (101 MHz;
DMSO-d6) d: 170.3 (C]O), 162.6 (d, J ¼ 239 Hz, Ar–C), 156.1
(C]O), 141.4 (d, J ¼ 11 Hz, Ar–C), 130.7 (d, J ¼ 10 Hz, Ar–C),
115.3 (Ar–C), 109.9 (d, J ¼ 21 Hz, Ar–C), 106.3 (d, J ¼ 26 Hz, Ar–
C), 78.2 (C-But), 37.3 (CH2), 36.9 (CH2), 28.7 (3 ꢂ CH3).
General procedure for mixed anhydride coupling
To Boc-b-alanyl (1.2 equivalents) in dry THF, N-methyl-
morpholine (1.05 equivalents) was added and reaction mixture
was cooled to ꢀ10 ꢃC and then stirred for 30 min. Isobutyl
chloroformate (1.05 equivalent) was then added, the mixture
ꢃ
was stirred (2 min) and then a pre-cooled (ꢀ10 C) solution of
the appropriate aniline (1 equivalent) dissolved in THF (8 mL)
ꢃ
was added drop-wise. The mixture was stirred (2 h) at ꢀ10 C,
allowed to warm to room temperature and then stirred over-
night. The mixture was concentrated and water was added to
the residue. The mixture was extracted with CH2Cl2 (2 ꢂ 20 mL)
and the combined organic extracts were washed sequentially
with aqueous citric acid solution (0.1 M, 2 ꢂ 20 mL), aqueous
NaHCO3 solution (10%, 2 ꢂ 20 mL), water (2 ꢂ 20 mL), dried
(MgSO4) and concentrated to give the crude product. Recrys-
tallisation from methanol gave pure compounds as white
crystals.
Results & discussion
Analytical data was determined for all three tested exogenous
volatile organic compound standards i.e. aniline, 3-uoroani-
line and p-toluidine, by HS-SPME-GC-MS. Calibration graphs
were also obtained for all three VOCs over the concentration
range 0–100 mg mLꢀ1 with correlation coefficients, R2, of >0.99
in all cases. The limit of detection (LOD) and limit of quanti-
tation (LOQ), based on 3 or 10ꢂ standard deviations of the
blank, were determined for each VOC (Table 1). The inclusion of
a pre-concentration step, using SPME, allowed excellent LOD
General procedure for Boc-deprotection
The appropriate Boc-protected substrate 2a–c was suspended in and LOQ to be obtained using GC-MS (Table 1), typically 0.02 mg
dry CH2Cl2 and then triuoroacetic acid was added dropwise. mLꢀ1 and 0.06 mg mLꢀ1 for 3-uoroaniline.
The resulting mixture was stirred (4 h) at room temperature and
Initially, the three potential enzyme substrates were tested
evaporated. The resulting oil was triturated with diethyl ether against a panel of 12 P. aeruginosa strains and 12 Burkholderia
until a precipitate formed. The solid was collected and washed cepacia complex strains (Table 2). All bacteria were tested in BHI
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Table 1 Analytical data for the quantification of exogenous volatile organic compounds by HS-SPME-GC-MS for analysis of sputum samples
from cystic fibrosis patients
GC retention
time (mins)
Linear range
LOD (mg
LOQ (mg
VOC
Quantitative m/z
(mg mLꢀ1
)
y ¼ mx + c
R2
n
mLꢀ1
)
mLꢀ1
)
Aniline
6.2
6.7
7.6
39–66–93
83–84–111
77–106–107
0–100
0–100
0–100
y ¼ 5146.7 + 83.4
y ¼ 18 337x + 314.2
y ¼ 11 163x + 417.3
0.9970
0.9970
0.9970
5
5
5
0.15
0.02
0.09
0.37
0.06
0.24
3-Fluoroanilinea
p-Toluidine
a
Three 85 mm polyacrylate SPME bres were used during the analysis of cystic brosis patient sputum samples. Previous use of SPME technology
identied that bre degradation does not occur until around 200 injections; in this study SPME bres were used for a maximum of 125 injections to
ensure consistency between samples. Due to variability of adsorptive efficiency within the SPME, a new calibration for 3-uoroaniline was generated
each time a new bre was used; in addition, the LOD and LOQ was also re-calculated for each bre. The analytical data obtained was as follows:
SPME bre 1: y ¼ 18 694x + 308.5, R2 ¼ 0.9970, LOD 0.017 mg mLꢀ1 and LOQ ¼ 0.057 mg mL; SPME bre 2: y ¼ 18 374x + 254.9, R2 ¼ 0.9960, LOD
0.017 mg mLꢀ1 and LOQ ¼ 0.057 mg mL; and, SPME bre 3: y ¼ 18 136x + 267, R2 ¼ 0.9970, LOD 0.015 mg mLꢀ1 and LOQ ¼ 0.049 mg mLꢀ1
.
broth at a pre-incubation inoculum of 1 ꢂ 106 CFU mLꢀ1, and signicant difference between the mean values of enzyme
substrates were added at 100 mg mLꢀ1, equivalent to 1.07 mmol activity, as assessed by the VOC concentration, between the 3
Lꢀ1 aniline, 1.11 mmol Lꢀ1 3-uoroaniline and 0.93 mmol Lꢀ1 substrates. However, an ANOVA does not give an indication to
p-toluidine, using NMP as the solvent. All samples were incu- where the difference between the groups lies, and therefore
bated overnight at 37 ꢃC prior to sampling via HS-SPME-GC-MS. requires individual T-tests between paired groups to determine
Good activity was observed (typically > 20 mg mLꢀ1 VOC liber- which substrate produced the best results. Prior to T-tests, F-
ated) for all P. aeruginosa strains across all 3 b-alanyl amino- tests were performed to determine equal or unequal variance
peptidase substrates. This supports the previous ndings in the between the paired sample groups. An initial F-test was con-
literature regarding activity in this species.29,30 There was some ducted between the data from substrates 3-amino-N-(3-uo-
variation in enzyme activity between the P. aeruginosa strains, rophenyl)propanamide
and
3-amino-N-(4-methylphenyl)
for instance using substrate 3-amino-N-(3-uorophenyl)prop- propanamide, which gave an F value of 17.4, and an F critical
anamide the AA44 strain produced an average of 38.5 mg mLꢀ1 value of 2.8; as the F value was greater than the F critical value,
(or 346 mmol Lꢀ1) of its VOC product (3-uoroaniline), whereas we can reject the null hypothesis that sample variance is equal
strain AUS52 produced an average of 19.8 mg mLꢀ1 (or 178 mmol between these datasets, and accept the alternative hypothesis
Lꢀ1). In general terms, the 3-amino-N-(4-methylphenyl)prop- that the variance is unequal. Next, an F-test was carried out for
anamide substrate evolved its VOC (i.e. p-toluidine) almost the data acquired from 3-amino-N-(3-uorophenyl)prop-
always to a lower concentration (Table 2).
anamide and 3-amino-N-phenylpropanamide, giving subse-
Enzyme activity was markedly less substantial in the B. quent F and F critical values of 2.2 and 2.8, respectively,
cepacia complex strains than in P. aeruginosa but was still therefore indicating equal variance between the two datasets.
signicant enough for straightforward quantication. B. multi- Finally, another F-test was conducted between substrates 3-
vorans, B. cenocepacia, B. cepacia, and B. stabilis all gave positive amino-N-(4-methylphenyl)propanamide and 3-amino-N-phe-
results for b-alanyl aminopeptidase activity, with only B. viet- nylpropanamide, giving subsequent F and F critical values of 7.8
namiensis testing negative in LMG strains 10 929 and 16 232 and 2.8, respectively, therefore indicating unequal variance
across all substrates. As was observed in the P. aeruginosa between the two datasets.
strains, there was signicant variability in enzyme activity
Following the implementation of F-test analyses, T-test
between members of the same species when using the same analyses were then performed on paired data sets. Firstly, a T-
substrate. For instance, B. multivorans was the most tested test assuming unequal sample variances was conducted
member of the B. cepacia complex strains with 5 strains tested, between 3-amino-N-(3-uorophenyl)propanamide and 3-amino-
and when tested with substrate 3-amino-N-(3-uorophenyl) N-(4-methylphenyl)propanamide, giving a two tail p-value of
propanamide produced average VOC concentrations of between 0.0009 meaning there is a statistically signicant difference (p <
1.2 mg mLꢀ1 and 12.8 mg mLꢀ1. Overall, the results from the b- 0.05) between the means of the two datasets, with 3-amino-N-(3-
alanyl substrates were deemed to be acceptable (Table 2) despite uorophenyl)propanamide producing a higher concentration
the lack of activity in B. vietnamiensis, however this is somewhat of VOC product from enzyme activity. Next a T-test assuming
offset given the minimal role B. vietnamiensis plays in cystic equal sample variance was performed between 3-amino-N-(3-
brosis associated infections in comparison to B. multivorans, uorophenyl)propanamide
and
3-amino-N-phenyl-
B. cenocepacia, and B. cepacia.37
propanamide, gave a two tail p-value of 0.3350 (p < 0.05),
In order to ascertain which substrate would be most suitable meaning that no statistically signicant difference can be
for further testing, a statistical analysis on the performance of inferred between these two datasets, and that the substrates
each b-alanyl substrate amongst the P. aeruginosa strains was behave in a very similar manner in terms of VOC concentration
performed. A single factor ANOVA, with a p-value of 0.0002 (p # evolution. Lastly, a T-test assuming unequal samples variances
0.05) was performed which indicated there was a statistically was carried out between 3-amino-N-(4-methylphenyl)
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Table 2 Preliminary b-alanyl substrate testing of P. aeruginosa strains and B. cepacia complex strains
b-Alanyl substrates
Substrate: 3-amino-N-(3-
Substrate: 3-amino-N-(4-
Substrate: 3-amino-N-
uorophenyl)propanamide
methylphenyl) propanamide
phenylpropanamide
VOC: 3-uoroaniline
VOC: p-toluidine
VOC: aniline
ID number
Strain ID
Growth
mg mLꢀ1
Growth
mg mLꢀ1
Growth
mg mLꢀ1
LMG 27632
LMG 27631
LMG 27629
LMG 27638
LMG 27626
LMG 27624
LMG 27627
LMG 27630
LMG 27622
LMG 27625
LMG 27623
NCTC 12903
LMG 18824
LMG 14294
LMG 18828
LMG 18822
LMG 18821
LMG 16656
LMG 10929
LMG 17588
LMG 16660
LMG 13010
LMG 16232
LMG 1222
P. aeruginosa AA44
P. aeruginosa AA43
P. aeruginosa AUS52
P. aeruginosa PA01
P. aeruginosa DK2
P. aeruginosa LES431
P. aeruginosa AES1R
P. aeruginosa AA2
P. aeruginosa LESB58
P. aeruginosa C3719
P. aeruginosa LES400
P. aeruginosa NCTC 12903
B. multivorans
B. stabilis
B. cenocepacia
B. multivorans
B. cepacia
B. cenocepacia
B. vietnamiensis
B. multivorans
B. multivorans
B. multivorans
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
38.5 (37.6, 39.3)
33.4 (34.5, 32.4)
19.8 (19.8, 19.7)
32.7 (32.3, 33.1)
35.6 (35.4, 35.7)
35.5 (36.3, 34.8)
26.4 (25.6, 27.2)
26.0 (25.9, 26.1)
24.9 (24.7, 25.1)
23.9 (23.4, 24.3)
20.7 (20.8, 20.6)
21.2 (20.7, 21.2)
1.2 (1.2, 1.2)
8.4 (8.2, 8.6)
2.3 (2.3, 2.2)
1.8 (1.8, 1.9)
2.4 (2.4, 2.4)
7.9 (8.1, 7.7)
0.0 (0.0, 0.0)
12.8 (12.5, 13.2)
1.5 (1.4, 1.5)
2.8 (2.8, 2.8)
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
21.0 (20.9, 21.2)
22.0 (21.4, 22.6)
20.8 (20.1, 21.5)
17.5 (17.5, 17.4)
21.3 (21.2, 21.5)
19.7 (19.3, 20.2)
19.5 (19.1, 20.0)
17.9 (18.1, 17.7)
21.1 (21.4, 20.7)
18.4 (18.9, 17.9)
18.0 (17.9, 18.2)
18.3 (17.9, 18.7)
2.5 (2.5, 2.5)
15.7 (15.4, 15.9)
4.4 (4.4, 4.4)
3.8 (3.9, 3.8)
4.1 (4.1, 4.2)
10.3 (10.4, 10.2)
0.0 (0.0, 0.0)
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
36.4 (37.1, 35.8)
27.3 (28.2, 26.5)
20.0 (20.3, 19.8)
28.6 (28.8, 28.5)
28.4 (27.4, 29.3)
26.5 (26.0, 26.9)
25.9 (26.4, 25.4)
22.9 (23.6, 22.2)
28.4 (27.7, 29.1)
22.5 (23.0, 21.9)
22.1 (21.8, 22.4)
22.6 (23.1, 22.1)
1.0 (1.0, 1.0)
6.8 (6.6, 7.0)
2.6 (2.5, 2.6)
2.3 (2.3, 2.3)
2.2 (2.2, 2.1)
5.8 (5.8, 5.8)
0.0 (0.0, 0.0)
10.6 (10.8, 10.5)
2.3 (2.3, 2.3)
5.5 (5.2, 5.9)
21.0 (21.1, 20.9)
3.4 (3.3, 3.4)
8.9 (8.7, 9.2)
0.0 (0.0, 0.0)
5.0 (4.9, 5.1)
B. vietnamiensis
B. cepacia
0.0 (0.0, 0.0)
1.7 (1.7, 1.7)
0.0 (0.0, 0.0)
3.1 (3.0, 3.1)
propanamide and 3-amino-N-phenylpropanamide, which gave (Fig. 1(a)), with increasing VOC evolution observed aer 6 h of
a two tail p-value of 0.0003 (p < 0.05), meaning there was incubation in all tested strains; typical 3-uoroaniline concen-
a statistically signicant difference between the average VOC trations ranging from 1.2 mg mLꢀ1 to 4.6 mg mLꢀ1, which was at
concentration evolution in our 12 P. aeruginosa strains between least 20 times higher than the estimated LOQ (Table 1). Results
these two substrates, with 3-amino-N-phenylpropanamide the for the B. cepacia complex strains were understandably less
more productive of the two.
impressive than those of the P. aeruginosa strains. None of the 4
The results of all the T-test analyses indicated that whilst 3- strains were able to produce any VOC evolution following 6 h of
amino-N-(4-methylphenyl)-propanamide performed the poorest incubation, and only 2 of the 4 strains tested were able to
out of the three b-alanyl aminopeptidase substrates, there was produce a detectable VOC signal following 8 h of incubation (B.
no statistically signicant difference between the performance stabilis and B. cenocepacia). However, all 4 of the tested strains
of 3-amino-N-(3-uorophenyl)propanamide and 3-amino-N- were able to produce the VOC following 10 h of incubation;
phenylpropanamide. However, from the initial calibration data typical 3-uoroaniline concentrations ranging from 0.3 mg mLꢀ1
in Table 1, for the two VOCs produced by these compounds (3- to 2.2 mg mLꢀ1, which was at least 5 times higher than the
uoroaniline by 3-amino-N-(3-uorophenyl)propanamide, and estimated LOQ (Table 1).
aniline by 3-amino-N-phenylpropanamide), the HS-SPME-GC-
MS method gave a lower limit of detection and limit of quan- Detection of P. aeruginosa strains and B. cepacia complex
tication for 3-uoroaniline than aniline; therefore making 3- strains in sputum from patients with cystic brosis
amino-N-(3-uorophenyl)propanamide the most suitable
enzyme substrate for subsequent testing.
Subsequently a time study was performed using the enzyme
substrate, 3-amino-N-(3-uorophenyl)propanamide, to deter-
mine 3-uoroaniline evolution from a panel of 4 randomly
selected P. aeruginosa strains and 4 strains that represent the
sub-species of the B. cepacia complex. The results (Fig. 1), from
the time study on P. aeruginosa strains, were consistent
A blind study was undertaken of 100 sputum samples collected
and analysed over the period of one calendar month from 78
distinct CF patients (49 male : 51 female). In addition, a further
5 samples were analyzed from CF patients who were post-lung
transplant. All generated data was evaluated post-analyses
(Table 3). The data identied that no false negative samples
were observed in the sputum sample data set. However, there
were 7 instances in which initial consideration of the exogenous
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Fig. 1 Evolution of 3-fluoroaniline using the enzyme substrate 3-amino-N-(3-fluorophenyl)propanamide with selected (a) P. aeruginosa strains
and (b) B. cepacia complex strains.
VOC data concluded a false positive result based on identica- amino-N-(3-uorophenyl)propanamide and BHI broth as the
tion of b-alanyl aminopeptidase enzyme activity; this contra- growth medium, and all were re-conrmed to be positive for b-
dicted the initial culture – MALDI-TOF-MS data which had not alanyl aminopeptidase activity.
detected the presence of P. aeruginosa. However, further inves-
Respiratory infections caused by P. aeruginosa are very
tigation of these ‘false positive’ samples (samples 6, 13, 16, 17, common in patients with CF and once chronic infection is
27, 34 and 85) by further culturing, isolation and MALDI-TOF- established, it is almost impossible to eradicate and is associ-
MS conrmed the presence of a potentially b-alanyl amino- ated with increased mortality and morbidity.17 If infection can
peptidase positive organism that had been originally unre- be detected early, there is deemed to be a ‘window of opportu-
ported by the routine culture protocol for CF patient samples. nity’ for treatment with antibiotics that can result in eradication
The subsequent analyses identied the b-alanyl aminopepti- that is sustained for up to two years.17 Isolation of P. aeruginosa
dase activity as being attributable to Serratia marcescens (sample using standard culture methods is not always straightforward as
27), P. aeruginosa (samples 6, 16, 17 and 85), and a Pseudomonas the lung airways of patients with CF may contain several dozen
strain (sample 13 and 34); the latter could only be identied to distinct bacterial species and well as fungi38 that can compete
genus level via MALDI-TOF-MS. All organisms isolated from the with P. aeruginosa to form detectable colonies on culture media.
re-cultured samples were then tested as pure colonies using 3- Isolation of colonies by culture can be particularly challenging if
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Table 3 Comparison of exogenous volatile organic compound analysis by HS-SPME-GC-MS with culture – MALDI-TOF-MS of sputum samples
from cystic fibrosis patients
3-Fluoroaniline concentrationa (mg
mLꢀ1
)
Sample ID
number
Organisms identied from culture via
MALDI-TOF-MS
Patient gender
Patient age
8 h incubation
24 h incubation
Notes
b
1
2
3
4
5
6
M
F
F
F
F
51
10
9
9
9
Candida sp., P. uorescens, S. aureus
Candida sp., E. dermatitidis
E. dermatitidis, P. mirabilis
Candida sp., E. dermatitidis, P. mirabilis
A. fumigatus, E. dermatitidis, P. mirabilis
E. coli, E. dermatitidis, P. mirabilis, S.
maltophilia, P. aeruginosa
E. dermatitidis, P. mirabilis, S. maltophilia
A. fumigatus, Candida sp., E. dermatitidis,
P. mirabilis
3.0 (2.8, 3.2)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.5 (0.5, 0.5)
23.7 (22.9, 24.5)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.8 (0.8, 0.8)
c
F
9
7
8
F
F
9
9
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
9
10
11
M
F
F
17
11
11
A. fumigatus, Serratia sp.
A. fumigatus, E. dermatitidis
A. fumigatus, E. dermatitidis, P.
aeruginosa
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.3 (0.3, 0.3)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
33.7 (33.5, 33.9)
b
d
12
13
M
M
19
19
A. fumigatus, E. dermatitidis, M. abscessus
complex
A. fumigatus, Candida sp., E. dermatitidis,
Pseudomonas sp.
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.6 (0.7, 0.6)
2.5 (2.5, 2.6)
14
15
16
M
M
F
7
7
14
Candida sp.
0.0 (0.0, 0.0)
0.4 (0.4, 0.4)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
1.7 (1.7, 1.7)
0.4 (0.4, 0.4)
b
c
Candida sp., P. aeruginosa, Serratia sp.
Candida sp., S. apiospermum, P.
aeruginosa
S. apiospermum, P. aeruginosa
Candida sp., P. aeruginosa, S.
apiospermum
c
17
18
F
F
14
14
0.0 (0.0, 0.0)
5.1 (4.8, 5.4)
4.0 (3.9, 4.2)
8.0 (8.3, 7.8)
b
19
20
21
22
23
24
25
26
M
M
M
M
F
F
F
F
24
24
7
27
13
13
13
13
S. aureus
0.0 (0.0, 0.0)
0.6 (0.5, 0.6)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
1.5 (1.5, 1.5)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
b
A. fumigatus, P. aeruginosa, S. aureus
Candida sp., E. coli, E. dermatitidis
A. fumigatus, S. aureus, S. maltophilia
Achromobacter sp., E. dermatitidis
Achromobacter sp., E. dermatitidis
Achromobacter sp., E. dermatitidis
Achromobacter sp., Candida sp., E.
dermatitidis
Candida sp., E. miricola, E. dermatitidis, S.
marcescens, M. abscessus complex
Candida sp., E. dermatitidis
Candida sp.
Candida sp.
Candida sp., E. dermatitidis, Serratia sp.,
S. maltophilia
Candida sp., E. dermatitidis, Serratia sp.,
M. llatzerense
P. aeruginosa
e
27
M
10
0.0 (0.0, 0.0)
3.0 (3.0, 3.0)
28
29
30
31
M
M
M
F
10
10
10
16
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
1.7 (1.4, 1.9)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
4.8 (4.7, 5.0)
b
b
32
F
16
2.2 (2.0, 2.1)
3.7 (3.7, 3.6)
b
d
b
b
33
34
35
36
F
F
F
M
17
17
17
32
6.5 (7.0, 6.0)
8.7 (8.8, 8.6)
1.7 (1.6, 1.8)
0.4 (0.4, 0.4)
28.5 (26.6, 30.3)
14.8 (14.6, 15.1)
5.4 (5.2, 5.6)
Pseudomonas sp.
A. fumigatus, P. aeruginosa
B. multivorans, Candida sp., S. aureus, S.
maltophilia
2.0 (2.0, 2.1)
37
38
39
40
41
42
43
F
M
F
F
F
23
24
27
27
32
34
38
Pandoraea sp., M. abscessus complex
P. aeruginosa
P. aeruginosa
P. aeruginosa
P. aeruginosa, S. aureus
A. fumigatus, Candida sp., P. aeruginosa
A. fumigatus, B. multivorans, Candida sp.,
S. maltophilia, M. chelonae complex
Candida sp., P. aeruginosa
Candida sp., Pandoraea sp., P. aeruginosa
Achromobacter sp.
0.0 (0.0, 0.0)
2.2 (2.0, 2.4)
9.5 (8.9, 10.1)
4.0 (3.7, 4.3)
3.6 (3.6, 3.7)
0.3 (0.3, 0.3)
0.3 (0.3, 0.3)
0.0 (0.0, 0.0)
5.4 (5.2, 5.7)
17.9 (17.1, 18.7)
13.5 (13.5, 13.6)
7.1 (6.7, 7.4)
5.3 (5.6, 5.1)
0.7 (0.7, 0.7)
b
b
b
b
b
b
F
M
b
b
44
45
46
M
F
F
31
49
65
2.7 (2.6, 2.9)
0.3 (0.3, 0.3)
0.0 (0.0, 0.0)
22.6 (21.8, 23.4)
0.5 (0.5, 0.5)
0.0 (0.0, 0.0)
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Table 3 (Contd.)
3-Fluoroaniline concentrationa (mg
mLꢀ1
)
Sample ID
Organisms identied from culture via
number
Patient gender
Patient age
MALDI-TOF-MS
8 h incubation
24 h incubation
Notes
b
b
47
48
M
M
33
31
Candida sp., P. aeruginosa, S. maltophilia
B. multivorans, Candida sp., P.
aeruginosa
2.5 (2.3, 2.6)
2.8 (2.7, 3.0)
5.1 (5.0, 5.2)
4.8 (4.5, 5.1)
49
50
51
F
F
M
8
24
29
N/A
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
2.9 (2.9, 2.9)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
3.7 (3.7, 3.7)
Candida sp., S. agalactiae (GpB)
E. coli, P. aeruginosa, M. llatzerense, M.
chelonae complex
E. dermatitidis, P. aeruginosa, M.
abscessus complex
b
b
b
52
53
M
M
22
35
0.3 (0.3, 0.4)
0.4 (0.3, 0.4)
0.5 (0.5, 0.5)
1.0 (0.9, 1.1)
B. multivorans, Candida sp., P.
aeruginosa
54
55
56
57
58
59
60
M
F
M
F
M
F
M
8
Neisseria elongata
A. fumigatus, S. aureus
Candida sp., P. aeruginosa
Achromobacter sp., P. aeruginosa
N/A
P. aeruginosa
A. fumigatus, Candida sp., P. aeruginosa,
S. aureus
Candida sp., P. aeruginosa, M. llatzerense
P. aeruginosa
P. aeruginosa
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
7.8 (7.9, 7.8)
4.7 (4.4, 5.0)
0.0 (0.0, 0.0)
13.5 (12.8, 14.1)
0.4 (0.3, 0.4)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
9.3 (9.2, 9.4)
8.2 (8.0, 8.4)
0.0 (0.0, 0.0)
27.9 (28.5, 27.2)
0.7 (0.7, 0.7)
19
29
30
62
46
42
b
b
b
b
b
b
b
61
62
63
64
65
66
67
68
69
70
F
F
F
M
M
M
M
F
40
44
14
49
35
38
12
48
36
24
5.7 (5.5, 5.9)
4.0 (3.8, 4.3)
14.6 (14.9, 14.4)
0.0 (0.0, 0.0)
8.2 (8.1, 8.3)
4.3 (4.2, 4.3)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
6.7 (6.6, 6.9)
0.0 (0.0, 0.0)
21.0 (20.6, 21.4)
10.9 (10.9, 10.9)
22.3 (21.8, 22.7)
0.0 (0.0, 0.0)
25.4 (25.8, 25.0)
17.0 (16.6, 17.4)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
21.8 (22.1, 21.5)
0.0 (0.0, 0.0)
A. fumigatus, Candida sp., S. maltophilia
A. fumigatus, Candida sp., P. aeruginosa
P. aeruginosa
b
b
N/A
Candida sp., S. aureus
b
M
F
Candida sp., P. aeruginosa
A. fumigatus, E. dermatitidis, H.
inuenzae, S. aureus
P. aeruginosa, M. abscessus complex
Candida sp., P. aeruginosa, S.
maltophilia, M. abscessus complex
E. dermatitidis, P. aeruginosa
Candida sp., S. aureus
b
b
71
72
M
M
19
23
6.3 (6.3, 6.2)
0.6 (0.6, 0.6)
9.1 (8.8, 9.5)
1.1 (1.1, 1.1)
b
b
73
74
75
M
M
F
31
34
14
12.7 (12.9, 12.5)
0.0 (0.0, 0.0)
0.4 (0.4, 0.4)
22.5 (22.7, 22.3)
0.0 (0.0, 0.0)
1.0 (1.1, 1.0)
A. fumigatus, Candida sp., E. dermatitidis,
P. aeruginosa
76
77
M
M
37
29
Achromobacter sp.
Candida sp., E. dermatitidis, Rothia sp., S.
maltophilia
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
b
b
78
79
F
M
70
25
Candida sp., P. aeruginosa, S. aureus
Candida sp., E. dermatitidis, P.
aeruginosa, Rothia sp.
P. aeruginosa, M. abscessus complex
Achromobacter sp., Candida sp., P.
aeruginosa
0.3 (0.3, 0.3)
0.3 (0.3, 0.3)
0.5 (0.6, 0.5)
5.5 (5.4, 5.7)
b
b
80
81
M
F
24
21
0.9 (0.9, 1.0)
0.5 (0.5, 0.5)
25.0 (24.6, 25.5)
1.2 (1.1, 1.2)
b
b
b
c
82
83
84
F
F
F
27
35
39
Candida sp., H. inuenzae, P. aeruginosa,
Serratia sp., S. aureus
Candida sp., K. oxytoca, P. aeruginosa, S.
aureus
B. multivorans, Candida sp., S.
maltophilia
P. aeruginosa
A. fumigatus, S. aureus
Candida sp., S. aureus
Candida sp., S. aureus
A. fumigatus, S. maltophilia, M. abscessus
complex
2.7 (2.6, 2.8)
0.3 (0.3, 0.3)
0.3 (0.3, 0.4)
4.6 (4.5, 4.7)
1.2 (1.1, 1.2)
0.7 (0.7, 0.8)
85
86
87
88
89
M
F
F
M
M
9
24.5 (24.7, 24.2)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
32.9 (34.1, 31.7)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
26
66
32
47
b
90
F
18
A. fumigatus, Candida sp., P. aeruginosa
4.7 (4.6, 4.8)
24.4 (33.9, 34.8)
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Table 3 (Contd.)
3-Fluoroaniline concentrationa (mg
mLꢀ1
)
Sample ID
Organisms identied from culture via
number
Patient gender
Patient age
21
MALDI-TOF-MS
8 h incubation
24 h incubation
Notes
b
91
M
B. multivorans, Candida sp., P.
aeruginosa, Serratia sp.
Candida sp., P. aeruginosa
Candida sp., P. aeruginosa
Candida sp., S. aureus
Candida sp., P. aeruginosa
S. aureus
Candida sp., P. aeruginosa
Candida sp., P. aeruginosa
Achromobacter sp.
2.1 (2.1, 2.1)
8.5 (7.0, 9.9)
b
b
92
93
94
95
96
97
98
99
100
101
102
103
F
F
F
M
M
F
M
M
M
F
25
26
49
35
34
22
47
NR
NR
18
38
37
2.7 (2.6, 2.8)
7.8 (7.6, 8.1)
0.0 (0.0, 0.0)
0.5 (0.4, 0.5)
0.0 (0.0, 0.0)
0.3 (0.3, 0.3)
1.9 (2.0, 1.7)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
7.1 (6.9, 7.4)
0.0 (0.0, 0.0)
17.0 (17.5, 16.5)
4.5 (4.3, 4.7)
8.3 (7.9, 8.7)
0.0 (0.0, 0.0)
1.3 (1.4, 1.2)
0.0 (0.0, 0.0)
0.7 (0.7, 0.8)
3.8 (3.7, 4.0)
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
26.4 (26.9, 26.0)
0.0 (0.0, 0.0)
23.1 (26.2, 20.1)
b
b
b
Candida sp.
P. aeruginosa
b,f
f
M
F
Candida sp., S. aureus
Achromobacter sp., A. fumigatus, P.
aeruginosa
Achromobacter sp., P. aeruginosa
Candida sp., S. aureus
b,f
b,f
104
105
M
F
28
20
6.1 (6.5, 5.8)
0.0 (0.0, 0.0)
8.4 (8.7, 8.1)
0.0 (0.0, 0.0)
f
a
Mean (individual value). b b-Alanine activity detected + causative organism identied in routine culture testing. c P. aeruginosa initially missed by
d
routine culture; positive b-alanine activity determined following subsequent pure isolate testing. Pseudomonas sp. initially missed by routine
culture; positive b-alanine activity determined following subsequent pure isolate testing. S. marcescens initially missed by routine culture;
e
f
positive b-alanine activity determined following subsequent pure isolate testing. Post lung transplant sample. NR ¼ not recorded. NA ¼ not
applicable; no organisms detected.
the abundance of P. aeruginosa is very low, relative to other co- with standard culture methods, this new culture method
inhabitants of the lung. This is likely to be the case when P. demonstrated 100% sensitivity and resulted in the detection of
aeruginosa rst colonizes the airways prior to chronic coloni- six additional isolates of Pseudomonas species (four of which
zation and infection. To circumvent this problem, Billard- were conrmed as P. aeruginosa) that were initially missed by
Pomares et al. developed a quantitative polymerase chain culture. The very high negative predictive value (100% in this
reaction (PCR) method to detect P. aeruginosa in sputum study) means that such an assay could be appropriate as
samples and demonstrated a detection limit of around 15 CFU a screening technique for patients who are not yet colonized by
mLꢀ1 of sputum.39 Using this highly-sensitive method, they this pathogen. Future work will focus on a simplied means for
were able to demonstrate detection of P. aeruginosa in 10/33 detection of the VOC liberated by b-alanyl aminopeptidase
sputum samples that were negative using standard culture activity (e.g. using a colorimetric indicator) to obviate the
methods. They concluded that this method may be a very useful requirement for sophisticated instrumentation.
tool in association with bacterial culture for the diagnosis of
early P. aeruginosa colonization and the monitoring of P. aeru-
Conflicts of interest
ginosa eradication aer antimicrobial therapy in young CF
patients.39 One limitation of PCR methods, as highlighted by
There are no conicts of interest.
the authors, was the possibility that dead or non-viable P. aer-
uginosa would be detected and this could be particularly prob-
lematic if PCR is used to monitor the outcome of eradication
therapy – which is another scenario where the bacterial load of
Acknowledgements
P. aeruginosa may be low and more challenging to detect using
The authors acknowledge nancial support from Northumbria
standard culture methods.
´
University and bioMerieux. High resolution mass spectrometry
was acquired at the EPSRC UK National Mass Spectrometry
Facility at Swansea University.
Conclusions
To the best of our knowledge, this is the rst demonstration of
detection of P. aeruginosa by use of a custom-designed substrate
to liberate a detectable VOC. The test includes a selective
enrichment broth that can potentially accommodate a high
volume of sputum resulting in high sensitivity. In comparison,
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