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Journal of Microbiological Methods 111 (2015) 18

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Journal of Microbiological Methods


journal homepage: www.elsevier.com/locate/jmicmeth

A method for the detection of antibiotic resistance markers in clinical


strains of Escherichia coli using MALDI mass spectrometry
Philippa J. Hart a,1, Emmanuel Wey b,1, Timothy D. McHugh c, Indran Balakrishnan b, Omar Belgacem a,
a
Shimadzu, Wharfside, Trafford Wharf Road, Manchester M17 1GP, UK
b
Royal Free Hospital NHS Foundation Trust, UK
c
UCL Centre for Clinical Microbiology, Division of Infection and Immunity, UK

a r t i c l e i n f o a b s t r a c t

Article history: Matrix-assisted laser-desorption/ionisation time-of-ight mass spectrometry (MALDI-TOF MS) is one of the
Received 8 October 2014 most widely used mass spectrometry based approaches for bacterial identication and classication. The
Received in revised form 23 January 2015 relatively simple sample preparation requirements and the speed of analysis which can usually be completed
Accepted 24 January 2015
within a few minutes have resulted in the adoption and assimilation of MALDI-TOF MS into the routine diagnostic
Available online 26 January 2015
workow of Clinical microbiology laboratories worldwide. This study describes the facilitation of bacterial
Keywords:
discrimination based on antibiotic resistance markers through the implementation of MALDI-TOF MS. The
MALDI-TOF MS periplasmic compartment of whole bacterial cells contains several proteins which confer antibiotic resistance
Bacterial identication in the Enterobacteriaceae. In order to reduce the complexity of the sample to be analysed via MALDI-TOF MS,
Antibiotic resistance the periplasm was extracted and subjected to in solution tryptic digestion followed by nano-LC separation.
This method, established that peptide sequence biomarkers from several classes of antibiotic resistance proteins
could be predicted using protein/peptide database tools such as Mascot. Biomarkers for a CTX-M-1 group extend-
ed spectrum -lactamase, CMY-2 an Amp-C -lactamase, VIM a metallo--lactamase, TEM a -lactamase and
KanR an aminoglycoside modifying enzyme were detected. This allowed for discrimination at a species level
and at an almost identical strain level where the only difference between strains was the carriage of a
modied antibiotic resistance carrying plasmid. This method also was able to detect some of these biomarkers
in clinical strains where multiple resistance mechanisms were present.
2015 Elsevier B.V. All rights reserved.

1. Introduction and Ross, 1996; Claydon et al., 1996). Since the commercialization of
MALDI mass spectrometers dedicated to the analysis of micro-
The use of matrix assisted laser desorption ionisation (MALDI) mass organisms for research and diagnostic purposes, the amount of work
spectrometry (MS) for the analysis of biomolecules is not new (Tanaka being carried out in this eld has increased considerably. It is important
et al., 1988; Karas et al., 1987). MALDI is an ionisation technique that can to state, that at the moment the only accepted mass spectrometry (MS)
be used in combination with several mass analysers but is particularly based microbial identication technique is the so-called MALDI-TOF
well suited to time of ight analysers due to its pulsed nature (laser Library-based approach (Sandrin et al., 2013). This technique has
pulses). This technique can be a method of choice for the analysis of been accepted in clinical microbiology laboratories internationally.
peptides, larger proteins, carbohydrates, lipids and several other classes MALDI-TOF MS is an especially favourable mass spectrometry based
of compounds (Hillenkamp and Peter-Katalini, 2014). approach in the investigation and speciation of microorganisms. This is
In 1975, Catherine Fenselau's Group reported that Gram negative largely due to the rapid speed of analysis, often minimal sample prepa-
bacteria could be analysed using a mass spectrometer (Anhalt and ration and generally low costs associated with the technique (Emonet
Fenselau, 1975). In 1996, the use of MALDI for analysis of bacteria et al., 2010; Fagerquist et al., 2010 and Sandrin et al., 2013). This trend
started to raise interest as a method of choice for the detection and appears to be continuing in the realms of microbial strain typing and
characterization of pathogens (Holland et al., 1996; Krishnamurthy antibiotic resistance research. Escherichia coli is widely investigated
using MALDI-TOF-MS, sometimes this is through the use of a library
based approach, as described by Siegrist et al. (2007), where MALDI-
Abbreviations: MALDI, matrix assisted laser desorption ionisation; TOF, time of ight; TOF-MS was investigated for its ability to differentiate between similar
MS, mass spectrometry; LC, liquid chromatography; -CHCA, alpha-cyano-4- strains of E. coli from varying sources. MALDI-TOF MS analysis of E. coli
hydrocinnamic acid; TFA, triuoroacetic acid
Corresponding author.
strains has also been performed using alternative bioinformatics ap-
E-mail address: omar.belgacem@kratos.co.uk (O. Belgacem). proaches, as in the case of Fagerquist et al. (2010), where Top-down
1
Both authors contributed equally to the work. proteomics was used to distinguish pathogenic and non-pathogenic

http://dx.doi.org/10.1016/j.mimet.2015.01.020
0167-7012/ 2015 Elsevier B.V. All rights reserved.
2 P.J. Hart et al. / Journal of Microbiological Methods 111 (2015) 18

strains of E. coli. E. coli is of course, not alone in its investigation by The ammonium dihydrogen phosphate (ADHP), dithiothreitol (DTT),
MALDI-TOF MS, this has been extended to many other microbial iodoacetamide (IAA), acetonitrile (MeCN), Ethanol (EtOH),
species, such as: Yersinia enterocolitica, Pseudomonas spp., and Legionella triuoroacetic acid (TFA) and ammonium bicarbonate were purchased
spp. (Ochoa and Harrington, 2005; Hotta et al., 2010; Fujinami et al., from Sigma-Aldrich (Dorset, UK). PeriPreps Periplasting Kit (an
2011). Epicentre product, supplied by Cambio, Cambridge, UK) was used in
The emergence of multi-drug resistant (MDR) Enterobacteriaceae the extraction of periplasmic proteins. Porcine derived trypsin, used
that can produce Extended Spectrum -lactamases (ESBLs) is currently for digestion, was purchased in lyophilised form, from Promega
of major worldwide concern. By hydrolytic inactivation, they confer (Southampton, UK).
resistance to a wide spectrum of -lactam antibiotics currently used as
1st line empirical therapy in the management of Gram negative bacteri-
2.2. Bacterial strains and growth conditions
al infections. E. coli is the most frequent Gram-negative bacillus isolated
from blood cultures in clinical settings and CTX-M-15 bla gene the most
DH5 E. coli with varying constructs of the pBAD (pBR322 derived)
common coding for an ESBL worldwide (Hawkey and Jones, 2009;
plasmid were provided by courtesy of Prof. Neil Woodford at AMRML,
Livermore and Hawkey, 2005; Woodford et al., 2004).
PHE, Colindale, United Kingdom (previously HPA Colindale) as follows:
Therapeutic options to treat infections caused by these organisms
DH5 with no plasmid (Strain C), DH5 with pBAD harbouring TEM-1
have become severely limited and are often not included in empiric
-lactamase (Strain A), and DH5 with pBAD harbouring TEM-1, Kan-R
therapy. Carbapenems, which are the mainstay of therapy for ESBL-
and blaCTX-M-15 ESBL (Strain B). Clinical strains (from blood and
producers (Woodford et al., 2006), are now under threat because of
urine samples), of dened phenotypes and genotypes were obtained
the emergence and international dissemination of organisms producing
from the Biobank facility in the Department of Medical Microbiology
a sub-group of -lactamases called carbapenemases. These enzymes
at the Royal Free Hospital NHS Foundation Trust. Genotyping of strains;
hydrolyse and inactivate carbapenems, and have been recognised by
050679, 174718, and 169030 was performed using validated in-house
the WHO as a worldwide threat (WHO, Antimicrobial Resistance:
molecular methods. Phenotypic sensitivity testing of strains to antimi-
Global report on surveillance, 2014). It is in this climate that several
crobial agents was performed using CLSI and EUCAST validated disc
research groups have been looking more closely at expanding the
diffusion and MIC determination methods. The three laboratory strains
diagnostic role of MALDI-TOF MS to accurately detect and describe
A, B and C were grown aerobically on Columbia blood agar and on Luria
these new and evolving resistance mechanisms.
Bertani (LB) medium with and without the addition of L-arabinose to
In a study by Schaumann et al. (2012), an attempt was made to
give a nal composition of 2% L-arabinose. The addition of L-arabinose
introduce the screening for resistance mechanisms alongside the
to the agar was performed to induce the pBAD vectors into over
routine MALDI-TOF MS species identication. This involved the acquisi-
expression of a constitutively expressed gene whose product was the
tion of mass spectra from protein extracts of bacterial samples and
resistance protein CTX-M-15.
across the m/z range 200012,000. However, this study concluded
The three clinical strains are dened as follows. Strain 050679 is an
that no reliable discrimination could be made between the spectra
uropathogenic strain isolated from the Urine of patient A (female)
obtained across this mass range for ESBL and non-ESBL producing
with an E. coli urinary tract infection prior to antibiotic therapy.
bacteria. Other recent studies (Hrabk et al., 2011 and Jung et al.,
Molecular testing using laboratory validated in-house molecular assays
2014) have taken an alternative approach and instead, focussed on the
and Reference laboratory molecular assays indicated the absence of
changes in the antibiotics themselves when added to bacterial strains
chromosomally or plasmid encoded resistance to amino-peniciliins,
possessing resistance to them. The study of Hrabk et al., investigated
quinolone class, aminoglycoside class and cephalosporin class antibi-
the activity of carbapenemase in multiple strains of bacteria and looked
otics. Strain 174718 was isolated in blood cultures of patient B (Female)
specically at the hydrolysis of Meropenem. Further experiments have
with an E. coli bacteraemia whilst being treated with Meropenem,
also been performed in this area, pertaining to the level of hydrolysis
Colistin and Tigecycline. Molecular testing using laboratory validated
of ampicillin when added to ESBL and non-ESBL producing bacteria
in-house molecular assays and Reference laboratory molecular assays
(Jung et al., 2014). In this study a larger number of isolates were
indicated the presence of blaNDM-1, blaCMY-2 (CIT-type AmpC gene),
analysed, however, the detection of resistance was still reliant on the
and blaSHV-28 on an IncA/C Plasmid. A 16S rRNA methylase gene
application of groups of antibiotics to the microorganisms, rather than
rmtC was also detected, conferring resistance to all clinically used
the direct analysis of the microorganisms themselves.
aminoglycosides. This strain was sensitive to Colistin and Tigecycline
In 2007 Russell et al., were able to facilitate the detection of plasmid
only.
insertion in E. coli, through the MALDI-TOF MS detection of -lactamase.
Strain 169030 was isolated in blood cultures of patient C (male) with
Russell et al., used in situ tryptic digestion to facilitate the identication
an E. coli bacteraemia whilst being treated with Meropenem and
of -lactamase in laboratory manipulated isolates. In our study, we have
Teicoplanin. Molecular testing using laboratory validated in-house
continued along a similar route to use a proteomics-based approach for
molecular assays and Reference laboratory molecular assays indicated
the differentiation between antibiotic sensitive E. coli and those in
the presence of blaVIM-1 on a class 1 Integron on an as yet untyped
possession of different mechanisms of resistance. Protein extracts
plasmid. This strain was sensitive to Colistin and Tigecycline only. All
from E. coli tested underwent in-solution tryptic digestion prior to the
three clinical strains were grown on both Columbia blood agar and on
nano-LCMALDI-MS approach. LC fractions were spotted onto a
LuriaBertani (LB) agar without the addition of L-arabinose.
MALDI target plate and analysed in MS and MS/MS mode to identify
the sequence tags of peptides belonging to the known antibiotic
resistance-associated proteins. This approach was successful in both 2.3. Bacteria cell culture and periplasmic extraction
laboratory transformed strains and clinical wild-type strains where
multiple resistance mechanisms were present. E. coli strains were inoculated on to LB agar with and without 2% LB
arabinose or Columbia blood agar and left to culture overnight. Fraction-
2. Materials and methods ation of Periplasmic proteins from E. coli was performed using the
Epicentre PeriPrepsTM Periplasting System. Briey, whole cells of
2.1. Materials E. coli were taken from the agar plate and inoculated into 50 L of
periplasting buffer before being left at room temperature for 5 min.
The MALDI matrix, alpha-cyano-4-hydrocinnamic acid (-CHCA) After a further 5 min, another 50 L of water was added and kept on
was purchased from Laser Bio Labs (Sophia-Antipolis Cedex, France). ice for 5 min. Samples underwent centrifugation at 13,000 rpm for
P.J. Hart et al. / Journal of Microbiological Methods 111 (2015) 18 3

2 min, after which the supernatant was removed and placed into fresh described previously. All strains were identied correctly to species
tubes. level with a high level of condence. As expected, it was not possible
to differentiate in a qualitative manner between the strains containing
2.4. Tryptic digestion the different plasmid constructs or those that were without plasmids.
The dendrogram in Fig. 1 was produced using SARAMIS software and
The periplasmic protein extracts were reduced through the addition compares samples of strains AC in quadruplicate. Samples are grouped
of 2.5 L of 45 mM dithiothreitol (DTT) (Sigma Aldrich) to 100 L vol- based on their relative spectral similarity.
umes of extract. The reduction was facilitated at 50 C for 15 min. The
samples were then alkylated through the addition of 2.5 of 10 mM 3.2. Resistance marker identication
iodoacetamide (IAA) (Sigma Aldrich), to each 100 volume of sample.
Alkylation was facilitated at room temperature in the dark for a further For the differentiation of the strains of E. coli analysed in this study a
15 min. Trypsin (Promega) was reconstituted at a concentration of new workow was initiated around the identication of resistance
20 g/mL in ammonium bicarbonate, and added to the reduced and markers that may be present with low overall abundance. Fig. 2,
alkylated sample at a ratio of 1:49. Digestion was facilitated at 37 C shows the steps of this workow from cultivation, through to database
overnight. searching. After cultivating the bacteria, periplasmic extraction was per-
formed on each of the individual strains analysed. This step was added
2.5. Nano-LCMALDI-MSMS to reduce the protein content and as such, complexity of the sample
prior to digestion. Subsequent to Tryptic digestion, LCMALDI MS and
Nano-LC was performed with the Chromolith CapRod trap (Merck), MS/MS were performed. Initially peptide mass ngerprints were gener-
RP-18e 50 0.2 mm (200 m ID), and the Chromolith CapRod column ated from each well/ fraction of the target plate. The LC-MALDI software
(Merck), RP-18e 150 0.1 mm (100 m ID). Two mobile phase (MP) used then automatically selected precursors from each of the peptide
solutions were used; MP A: 5% MeCN containing 0.05% TFA, and MP B: mass ngerprints on which to perform MS/MS (CID). For each of the
80/20 MeCN/H2O with 0.05% TFA. A 20 L volume of the sample was E. coli samples analysed N 500 MS/MS spectra were generated. All MS/
injected onto the column and a 60 minute gradient was run, at 035% MS results were then searched in MASCOT and matched to peptide
MP B. sequences.
Fractions from the LC were spotted onto the MALDI target plate at in-
tervals of 15 s, using the Accuspot (Shimadzu) automated spotter. Ma- 3.3. Resistance markers identied in laboratory strains
trix was deposited at the same time as the fractions, at a ow rate of 1
L/min. The matrix used was 5 mg/mL -CHCA in 70: 30 MeCN: H20, The resistance-associated proteins identied in each of the laborato-
0.1% TFA, 10 mM ADHP. ry strains are listed in Table 1. These included TEM-1 and CTX-M-1
Samples were analysed on an AXIMA Performance (Shimadzu, Man- group -lactamases, and Kan-R, an aminoglycoside modifying enzyme.
chester, UK), MALDI TOF-MS. Measurements were made in Reectron Also incorporated in this table is the number of peptide biomarkers
mode and across the mass range of 7003000 Da. A mixture of peptides, that were detected for each of the enzymes. Each of the peptides detect-
across the mass range 10462465 Da were used for calibration, which ed were matched to the NCBI database using MASCOT. Table 1, shows
was performed once for every 16 sample wells analysed. All MS/MS no results for the third laboratory strain analysed (C). This fully sensitive
were performed using high energy collision induced dissociation (HE- strain was used in order to act as a control, and as such no peptide bio-
CID). The MASCOT (Matrix Science, London, UK), search engine was markers belonging to resistance enzymes were detected. In addition to
used to search the NCBI database for protein/peptide matches. The tol- the resistance enzymes, many other periplasmic proteins/peptides
erances for MASCOT searches were set to 0.5 Da for precursor masses were identied in all strains. The presence or absence of ribosomal pro-
and 0.8 Da for MS/MS product ions. Only peptide matches with an ex- teins was used as a reference to determine the success of the periplas-
pected value of b 0.05 (b 5% chance of being false) were selected as pos- mic extract.
itive identications. For laboratory strain B, it was possible to increase the expression of
the CTX-M-15 protein by virtue of its presence on the pBAD vector by
2.6. MALDI-MS and SARAMIS adding L-arabinose to the LB agar. This resulted in over-expression and
an increased MIC to 3rd generation cephalosporins and Carbapenems
Pellets remaining from the periplasmic extraction described previ- (see Table 3). However, due to the sensitive nature of the LC-MALDI
ously were used to make the species level identication. A saturated method, the growth on Columbia blood agar or L-arabinose containing
matrix solution of -CHCA (40 mg/mL) in 33/33/33 MeCN/EtOH/H2O LB agar did not inuence the detection of the peptides that belonged
containing a nal concentration of 3% TFA was applied in a 1 L volume. to the CTX-M 1 group of enzymes (of which CTX-M-15 is a member).
Samples were then analysed on an AXIMA Performance (Shimadzu, Using the results from laboratory strain B, an example of a typical MS
Manchester, UK), MALDI TOF-MS. Measurements were made in linear spectrum obtained from the MALDI-TOF MS analysis of a single fraction
mode, across a mass range of 200020,000 Da. Subsequent to analysis subsequent to nano-LC is shown in Fig. 3(i). The peak at m/z 1567.04
all spectra were exported into SARAMIS (BioMerieux) software and was automatically selected as a precursor for MS/MS by the instrument
compared against the SARAMIS database (version 4.10). software, and the resulting CID spectrum can be seen in Fig. 3 (ii). The
MS/MS spectrum was compared to the NCBI database along with all
3. Results others from that experiment, using MASCOT. The MASCOT result for
this MS/MS spectrum is presented in Fig. 3 (iii), where the score is
3.1. Bacterial identication at the species level given and fragment ion series are clearly labelled.

All initial experiments were performed on laboratory strains of E. coli 3.4. Resistance markers identied in clinical isolates
in order to allow for a more controlled environment than is possible
when analysing patient samples. The three laboratory strains included: The resistance enzymes identied in each of the clinically isolated
DH5 with no plasmid (strain C), DH5 with pBAD harbouring TEM-1 - strains of E. coli are listed in Table 2. Also included in this table is the
lactamase (strain A), and DH5 with pBAD harbouring TEM-1, KanR number of peptide biomarkers that were detected for each of the en-
and blaCTX-M-15 ESBL (strain B). All samples were rst analysed in lin- zymes. As with the laboratory strains, each of the peptides detected
ear MALDI TOF-MS and identied using SARAMIS, via the workow were matched to the NCBI database using MASCOT. The clinical E. coli
4 P.J. Hart et al. / Journal of Microbiological Methods 111 (2015) 18

Fig. 1. A dendrogram produced using the SARAMIS software, comparing samples of strains AC in quadruplicate. Samples are grouped based on their relative spectral similarity.

strain 050679 was fully sensitive to antibiotics and carried no plasmids instruments being used and different operators preparing and analysing
harbouring -lactamases or aminoglycoside modifying enzymes. samples. The dendrogram in Fig. 1 shows that there is approximately
Hence, just as for the sensitive control (Strain C) in the laboratory 10% relative variation between each of the samples. The result was
manipulated group, no peptide biomarkers belonging to resistance deemed insignicant in this case, as the variation did not lead to the
enzymes were detected. correct grouping of the different strains.

4. Discussion 4.2. Resistance marker identication methodology

4.1. Species level bacterial identication E. coli whole cell material, as taken from agar plates is in essence a
very complex sample, containing proteins, carbohydrates, lipids, and
It was possible to identify the different strains (clinical and laboratory other biological molecules. Intact resistance enzymes which may differ-
manipulated) to the species level using the pellet remaining from the entiate the clinical strains in this study may have been of a molecular
periplasmic extraction and then following the conventional MALDI mass which is too high to be detected in such a complex sample (molec-
protein mass ngerprint/library based approach. This is to be expected, ular masses were veried through searching ExPASy (Gasteiger et al.,
as the pellet left over from the periplasmic extraction would contain a 2003 and Artimo et al., 2012)). When taking a whole cell approach a
large quantity of the ribosomal proteins that are typically present in large degree of ion suppression occurs, due to the presence of salts
the mass range used to make these species level identications and some particularly high abundance proteins (Cohen and Chait,
(Fenselau and Demirev, 2001). Strain level identication, or detection 1996; Kussmann et al., 1997). This may cause signals of interest to be
of resistance mechanisms using this whole cell, protein proling masked by others of high abundance or better response factor. Similar
approach was not found to be possible in this case. In a study by conclusions could be drawn from another study by Schaumann et al.
Arnold and Reilly (1998), the authors state that their correlation analysis (2012), where clinical strains of E. coli were analysed across an m/z
approach was only applicable where isolates were grown under strin- range of 200012,000. In this case the method used could not show
gent conditions and sample preparation was undertaken with great signicant discrimination between ESBL positive and ESBL negative
care ensuring spectral reproducibility. This would be very difcult to samples. There is some evidence to suggest that protein level discrimi-
maintain in clinical environments where there will be different nation may be possible when increasing the upper limit of the mass

Fig. 2. The LC-MALDI workow employed and examined in this study. Colonies were taken from agar plates, subject to periplasmic extraction and tryptic digestion before undergoing LC
separation. LC fractions were spotted onto a MALDI target plate in an automated manner, and analysed using MALDI-TOF MS and MS/MS. Finally, all MS/MS spectra were subject to a
protein/peptide database search using MASCOT.
P.J. Hart et al. / Journal of Microbiological Methods 111 (2015) 18 5

Table 1
Resistance-associated proteins detected in E. coli strains that were cultivated and manipulated in the laboratory. The peptide markers identied for each resistance enzyme are also given,
along with their sequences. Descriptions of the strains, AC are given in the Materials and Methods Section 2.2.

Resistance-associated protein Precursor detected [M+H]+ Laboratory strain Peptide sequences MASCOT score

TEM-1 1287.3645 A, B LLTGELLTLASR 42


1308.6094 VGYIELDLNSGK 53
1785.0212 IVVIYTTGSQATMDER 85
2023.3139 IHYSQNDLVEYSPVTEK 105
2084.5978 QQLIDWMEADKVAGPLLR 29
CTX-M-1 group 1287.3469 B SESEPNLLNQR 87
1350.5132 QLGDETFRLDR 43
1403.4977 ADERFAMCSTSK 47
1567.8461 LIAHVGGPASVTAFAR 113
1716.8996 APLILVTYFTQPQPK 99
1756.8375 SESEPNLLNQRVEIK 86
1838.0890 TGSGGYGTTNDIAVIWPK 113
1862.0912 LGVALINTADNSQILYR 187
1867.1915 LDRTEPTLNTAIPGDPR 85
1885.2471 KSESEPNLLNQRVEIK 31
1988.0399 DRAPLILVTYFTQPQPK 93
2140.5698 TEPTLNTAIPGDPRDTTSPR 80
2270.6304 VMAAAAVLKKSESEPNLLNQR 85
2525.1342 LDRTEPTLNTAIPGDPRDTTSPR 45
Kan-R 1059.0196 B FSGFIDCGR 56
1461.4520 DIAEELGGEWADR 89
1550.7796 FLVLYGIAAPDSQR 76
1968.3041 MIEQDGLHAGSPAAWVER 97
2741.1240 MPDGEDLVVTHGDACLPNIMVENGR 68
2871.1025 MEAGLVDQDDLDEEHQGLAPAELFAR 113
C

range to 30 kDa (Russell et al., 2007), but in strains where in-vitro cells (Curtis et al., 1972; Naglak and Wang, 1990 and Meini et al.,
plasmid transformation has been performed. In a study by Camara and 2013). By performing this extraction, molecules from other cellular
Hays, 2007, they detected an ion at m/z 29,000, present only in compartments, such as the highly abundant ribosomal proteins that
laboratory strains of E. coli known to possess ampicillin resistance. On are thought to be readily detectable via MALDI-MS analysis (Sauer and
this occasion, the identity of the marker peak was conrmed using Kliem, 2010), are removed. There still remain, however, a large number
in-gel digestion and subsequent peptide mass ngerprinting. However, of proteins within the periplasmic region of E. coli cells, making the extra
when attempting to apply this method to clinical strains, where lower nano-LC separation step subsequent to tryptic digestion important.
levels of resistance enzymes may be present (potentially due to the Through the use of nano-LC separation it was possible to drastically
presence of fewer copies of the plasmids bearing the genes that encode reduce the number of peptides per well, allowing for reduction of
the enzyme), these signals were not detected (Schaumann et al., 2012). overlapping peaks and suppression effects mentioned previously. This
As mentioned previously, when analysing complex samples using aided in the generation of good quality peptide sequences from MS/
MALDI-MS, suppression effects often caused by competitive desorp- MS (an example of which is shown in Fig. 3). As for the majority of
tion/ionisation in the gas phase are likely to be observed or experienced MS/MS spectra in this study, the main fragments in the MS/MS
(Cohen and Chait, 1996; Kussmann et al., 1997). This occurs when spectrum in Fig. 3 were found to correspond to y and b series ions. The
certain components of a sample desorb and/or ionise preferentially presence of ions from other series, indicating side chain fragmentation
over others, resulting in the alteration of signal intensity of specic was also observed in many cases. This is typical of spectra generated via
species and sometimes even the complete obscuration of said species the high energy CID implemented in this study (Belgacem et al., 2006
(Albrethsen, 2007; Montgomery et al., 2010). In an effort to reduce and Brgles et al., 2011).
ion suppression, extraction and separation steps were implemented,
prior and subsequent to tryptic digestion. 4.3. Resistance markers identied
Periplasmic extraction was chosen because the resistance markers
being searched for in this case (largely -lactamase enzymes) are Whilst it was possible to detect enough peptide biomarkers to con-
known to be located in the periplasmic compartment of microbial rm the presence of the CTX-M-1 group of -lactamase, the conserved

Table 2
Resistance-associated proteins detected in E. coli isolated and cultivated from clinical samples. The peptide markers identied for each resistance enzyme are also given, along with their
sequences.

Resistance-associated protein Precursor detected [M+H]+ Clinical strain Sequences MASCOT score

CMY-2 1291.4709 174718 TFNGVLGGDAIAR 57


1665.8913 LAHTWIYVPQNEQK 55
1704.8165 TGSTGGFGSYVAFVPEK 125
2082.4397 EGKPVHVSPGQLDAEAYGVK 95
2264.4485 LLHLATYTAGGLPLQIPDDVR 66
2370.4590 ADIANNHPVTQQTLFELGSVSK 82
2507.7223 LLHLATYTAGGLPLQIPDDVRDK 39
VIM-1 1252.2643 169030 AAGVATYASPSTR 17
2540.5878 LAEAEGNEIPTHSLEGLSSSGDAVR 114
050679
6 P.J. Hart et al. / Journal of Microbiological Methods 111 (2015) 18

Table 3 region of the protein sequence that would narrow the identication
A display of the minimum inhibitory concentration (MIC) values for the three laboratory down to the specic CTX-M enzyme (CTX-M-15), was not detected.
strains under varying growth conditions. CBA refers to Columbia blood agar and LB to
LuriaBertani agar.
This is more than likely due to the high level of homology within the
protein sequences of CTX-M-1 group -lactamases. When comparing
Strain Growth MIC to MIC to MIC to these sequences in ExPASy (Artimo et al., 2012) it is clear that CTX-M-
conditions amoxicillin mg/L ceftriaxone mg/L ertapenem mg/L
1 group members differ by the substitution of only a very small number
B CBA N8 4 b0.5 of amino acids, in some cases only one. The close evolutionary relation-
B LB N8 4 b0.5
ship of the CTX-M -lactamase enzymes is described in full by Novais
B LB with 2% N8 8 2
et al., 2010. The number of peptides detected for each enzyme in the
L-arabinose
A CBA N8 b1 b0.5 different strains varied. As shown in Table 1, it was possible to detect
A LB N8 b1 b0.5 as many as 15 peptides in one sample, but as little 2 in another
A LB with 2% N8 b1 b0.5 (which is today's accepted minimum). There are two plausible but not
L-arabinose mutually exclusive explanations for this. The rst being that theoretical
C CBA b8 b1 b0.5
digests indicate there to be fewer tryptic peptides generated for the
C LB b8 b1 b0.5
C LB with 2% b8 b1 b0.5
ambler class B metallobetalactamases (e.g. VIM-1, and NDM-1), within
L-arabinose
the mass range examined in this studied. Additionally, the matrix and
suppression effects as commented on previously, (which are inherent
to the MALDI as well as ESI method) might account for further reduction
in the number of peptides detected.

Fig. 3. An example of a typical MS spectrum acquired from a single fraction collected from a tryptic digest of the periplasmic extract of laboratory strain B (grown on Columbia blood agar)
(i). The MS/MS spectrum generated from the highlighted precursor ion for the -lactamase peptide at 1567 (ii). The corresponding MASCOT assignment of sequence and fragment ions
(iii). The precursor mass differs by m/z 0.49 between the MS and MS/MS spectra. This is to be expected as the ions in the MS spectrum are monoisotopically resolved and reported as such,
whereas the MS/MS spectrum is averaged and ions are not monoisotopically resolved.
P.J. Hart et al. / Journal of Microbiological Methods 111 (2015) 18 7

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