Monitoring bacterial resistance to chloramphenicol and other antibiotics by liquid chromatography electrospray ionization tandem mass spectrometry using selected reaction monitoring

Article abstract of DOI:10.1002/jms.3220

Antibiotic resistance is a growing problem worldwide. For this reason, clinical laboratories often determine the susceptibility of the bacterial isolate to a number of different antibiotics in order to establish the most effective antibiotic for treatment. Unfortunately, current susceptibility assays are time consuming. Antibiotic resistance often involves the chemical modification of an antibiotic to an inactive form by an enzyme expressed by the bacterium. Selected reaction monitoring (SRM) has the ability to quickly monitor and identify these chemical changes in an unprecedented time scale. In this work, we used SRM as a technique to determine the susceptibility of several different antibiotics to the chemically modifying enzymes β-lactamase and chloramphenicol acetyltransferase, enzymes used by bacteria to confer resistance to major classes of commonly used antibiotics. We also used this technique to directly monitor the effects of resistant bacteria grown in a broth containing a specific antibiotic. Because SRM is highly selective and can also identify chemical changes in a multitude of antibiotics in a single assay, SRM has the ability to detect organisms that are resistant to multiple antibiotics in a single assay. For these reasons, the use of SRM greatly reduces the time it takes to determine the susceptibility or resistance of an organism to a multitude of antibiotics by eliminating the time-consuming process found in other currently used methods. Copyright 2013 John Wiley & Sons, Ltd. Copyright

Full text of DOI:10.1002/jms.3220

Research article
Received: 24 January 2013
Revised: 9 April 2013
Accepted: 10 April 2013
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3220
Monitoring bacterial resistance to
chloramphenicol and other antibiotics
by liquid chromatography electrospray
ionization tandem mass spectrometry
using selected reaction monitoring
Anthony M. Haag,a* Audrie M. Medina,b Ariel E. Royall,b
Norbert K. Herzogc and David W. Nieselb
Antibiotic resistance is a growing problem worldwide. For this reason, clinical laboratories often determine the susceptibility
of the bacterial isolate to a number of different antibiotics in order to establish the most effective antibiotic for treatment.
Unfortunately, current susceptibility assays are time consuming. Antibiotic resistance often involves the chemical modification
of an antibiotic to an inactive form by an enzyme expressed by the bacterium. Selected reaction monitoring (SRM) has the ability
to quickly monitor and identify these chemical changes in an unprecedented time scale. In this work, we used SRM as a
technique to determine the susceptibility of several different antibiotics to the chemically modifying enzymes b-lactamase
and chloramphenicol acetyltransferase, enzymes used by bacteria to confer resistance to major classes of commonly used
antibiotics. We also used this technique to directly monitor the effects of resistant bacteria grown in a broth containing a
specific antibiotic. Because SRM is highly selective and can also identify chemical changes in a multitude of antibiotics in a
single assay, SRM has the ability to detect organisms that are resistant to multiple antibiotics in a single assay. For these reasons,
the use of SRM greatly reduces the time it takes to determine the susceptibility or resistance of an organism to a multitude of
antibiotics by eliminating the time-consuming process found in other currently used methods. Copyright © 2013 John Wiley &
Sons, Ltd.
Keywords: Selected Reaction Monitoring; Antibiotics; Resistance; Bacteria; Chloramphenicol
Susceptibility is determined from a standardized range of the
resulting diameter of inhibition of bacterial growth around the disk.
Conversely, the MIC dilution method is a quantitative method that
Introduction
Antibiotic resistance is a growing problem.[1] The overuse of
antibiotics and the adaptability of bacteria have created a growing
number of resistant bacterial strains.[2,3] In 2005, there were over
11,000 deaths from methicillin-resistant Staphylococcus aureus
alone.[4] Acenitobacter baumannii, an inherently multidrug resistant
organism, has recently emerged as one of the most common
infectious organisms of wounds in military service members
serving in Iraq and Afghanistan.[5–8] Enterococcus sp. have long
been associated with nosocomial infections and are often
multidrug resistant.[9] Because of the high mortality rate caused
by these organisms, rapidly determining which antibiotic an organ-
ism is susceptible to is imperative to improving clinical outcomes.
Current methods to determine the susceptibility or resistance
of bacteria to a particular antibiotic typically involves isolating
the organism from an individual specimen (blood, spinal fluid,
tissue, etc.). The isolated bacteria are grown on culture media,
and then one of two methods employed for analyzing antibiotic
resistance, Kirby–Bauer and minimal inhibitory concentration
dilution method (MIC). Kirby–Bauer is a qualitative method
whereby the bacteria to be tested are inoculated over the entire
surface of an agar plate. Then, disks containing a standardized
concentration of different antibiotics are placed on the plate.
allows determination of the lowest concentration of an antibiotic
that inhibits the visible growth of a microorganism. Although these
two methods are effective, susceptibility testing is a slow process,
often requiring 48–72 h for results.[10] Because of this delay, a
physician will often treat the patient with broad spectrum antibi-
otics until the susceptibility results are known. Treating with an
ineffective antibiotic not only may compromise the health of the
patient but also can contribute to the selection of antibiotic-
resistant organisms.[11] Therefore, it is necessary to develop a
*
Correspondence to: Anthony M. Haag, Biomolecular Resource Facility,
University of Texas Medical Branch, Galveston TX 77555, USA. E-mail:
amhaag@utmb.edu
a Biomolecular Resource Facility, University of Texas Medical Branch, Galveston,
TX 77555, USA
b Department of Microbiology and Immunology, University of Texas Medical
Branch, Galveston, TX 77555, USA
c
Department of Medical Sciences, Quinnipiac University, Hamden, CT 06518,
USA
J. Mass Spectrom. 2013, 48, 732–739
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Monitoring bacterial resistance by SRM
method to determine the organism’s susceptibility to numerous
antibiotics in as short of a time span as possible, thus preventing
increased resistance and also reducing the morbidity and mortality
among patients.
modification pathways used by the organism. Therefore, the
presence or absence of unmodified antibiotics by SRM could
determine whether an organism is expressing an antibiotic-
inactivating protein.
One common mechanism used by bacteria to enable their
resistance to antibiotics is the elaboration of enzymes that can
inactivate the antimicrobial activity of antibiotics by chemically
modifying their structure. b-lactamase, an enzyme expressed by
bacteria resistant to the b-lactam class of antibiotics, hydrolyzes
the b-lactam ring of these antibiotics, which include penicillins
and cephalosporins.[12,13] However, more recently approved
cephalosporins are less susceptible to hydrolysis by many types
of b-lactamase. Because of selective pressure on bacteria
through antibiotic use, mutations leading to structural changes
in the b-lactamase protein allow it to hydrolyze these newer
cephalosporins.[14] In the case of chloramphenicol, resistant
organisms produce chloramphenicol acetyltransferase that
acetylates the hydroxyl moiety, thereby preventing the drug from
binding to the bacterial ribosome.[15] Chemical modifications
can either reduce or eliminate the effectiveness of an antibiotic.
Therefore, methods to identify chemical changes to an antibiotic
allow one to immediately determine organism resistance to that
specific antibiotic. This is particularly true if an enzymatic
chemical modification is already known and has been correlated
to resistance by the organism.
Matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry (MALDI-TOF MS) has recently been reported as a
rapid technique for monitoring the chemical modifications of
antibiotics via enzymes expressed by antibiotic-resistant
organisms.[16–18] A number of different antibiotic-resistant organisms
were investigated including Escherichia coli, Klebsiella pneumoniae,
and Pseudomonas aeruginosa. Antibiotic resistance was detected
in these organisms by observing changes in the b-lactam
antibiotics, particularly hydrolysis. A number of different b-lactam
antibiotic were investigated including penicillins, cephalosporins,
and carbapenems. Although these results demonstrate the
effectiveness of MALDI-TOF MS as a qualitative technique for
determining antibiotic resistance, it suffers from being a superior
quantitative technique compared with other current mass spectro-
metric techniques used for quantitation, such as selected reaction
monitoring (SRM).
Materials and methods
Chemicals and reagents
Amoxicillin, ampicillin, nafcillin, cloxacillin, chloramphenicol, and
formic acid (FA) were purchased from Sigma-Aldrich (St. Louis,
MO). Dibasic sodium phosphate, monobasic potassium phosphate,
ammonium chloride, sodium chloride, magnesium sulfate, calcium
chloride, glucose, sodium azide, thiamine, and tryptophan were
purchased from Fischer (Waltham, MA). Casamino acids were
obtained from Difco (Sparks, MD). Ethanol was obtained from
Pharmco-AAPER (Brookfield, CT) and high-performance liquid
chromatography (HPLC) grade acetonitrile (ACN) from J.T
Baker-Mallinckrodt (St. Louis, MO). Water was obtained in-house
from a Milli-Q UF Plus filtration system and further purified by distilla-
tion. 3-O-acetylchloramphenicol and 1,3-O,O-diacetylchloramphenicol
were manufactured on campus by the University of Texas
Medical Branch (UTMB) Organic Synthesis Core facility.
Antibiotic stock solutions for HPLC optimization and quality
control were prepared by dissolving the corresponding antibiotic
in water and serially diluting to obtain a concentration of 500 ng/
ml. For cultures, ampicillin stock solution was prepared as a
50 mg/ml solution in water, and chloramphenicol stock solution
was prepared as a 30 mg/ml solution in ethanol. Because of its
low solubility, chloramphenicol was first dissolved in ethanol
(5 mg/ml) and then diluted to 500 ng/ml with water. Because of
the limited lifespan of b-lactam antibiotics in solution, stock
solutions were prepared daily.
M9 broth was prepared as follows: 5.3 g Na2HPO4, 3g KH2PO4, 0.5g
NaCl, 1.0g NH4Cl, and 2.5g casamino acids were dissolved in water
to a volume of 500 ml. The solution was autoclaved for 20 min, after
which 0.5ml 1M MgSO4 Á 7H2O, 0.1ml 0.5 M CaCl2, 2.5ml 40% (w/v)
glucose, 5 mg thiamine, and 10 mg of tryptophan were added.
Sample preparation
Selected reaction monitoring is a mass spectrometric technique
that has a long history of being able to detect compounds with
very high sensitivity and selectivity.[19,20] SRM has already been
used for the detection and quantification of antibiotics in the
human body, including the determination of cefuroxime in
plasma and the analysis of b-lactam antibiotics in kidney
tissue.[21,22] SRM has also been used to monitor the level of antibi-
otics in the environment, such as identifying and quantifying
antibiotics in waste water or determining the levels of tetracy-
cline in manure compost.[23,24] However, there has been little-
to-nothing published on the use of SRM for determining
antibiotic resistance.[25]
By monitoring the presence or absence of an antibiotic and/or
its chemically modified form after exposure to a bacterial culture,
SRM has the potential to be a rapid and accurate screening
technique for determining the susceptibility or resistance of such
bacterial organisms. This would allow one to screen the effective-
ness of numerous antibiotics to a particular bacterial organism in
a single assay. Moreover, the technique would not be limited to
antibiotic resistance based on a single chemical modification
pathway but rather also applicable to any known chemical
Ampicillin-resistant E. coli (pUC18) and chloramphenicol-resistant
E. coli (pACYC184) were separately cultured overnight in M9
broth. Overnight cultures (20 h) resulted in 1.0 Â 108 colony-
forming units/ml (cfu/ml). To 150 ml of fresh M9 broth, 250 ml of
pUC18-transformed E. coli and 15 ml of ampicillin stock solution
for culture were added. To another 150 ml of fresh M9 broth,
150 ml of pACYC184 transformed E. coli and 15 ml of chloramphenicol
stock solution for culture were added. The samples were then
mixed and incubated at 37 ꢀC. Next, 1.5 ml of broth was removed
each hour (including time = 0) and centrifuged at 3000 rpm. The
supernatant was removed, and 15 ml of 0.02% (w/v) NaN3 was
added. The supernatant solutions were then frozen (À80 ꢀC)
until ready for analysis by mass spectrometry.
Chromatography
HPLC separation was performed by using an LC Packings Nano-LC
system, consisting of an autosampler, a binary gradient pump, and
a loading pump. The column and trap column were made in-house.
The column was made from 75 mm ID polyimide-coated fused silica
capillary (Polymicro Technologies, Phoenix, AZ) packed with 5mm
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A. M. Haag et al.
Zorbax SB-C18 reversed-phase packing (Agilent, Santa Clara, CA) to
a length of 5 cm by using a Pressure Injection Cell (NextAdvance,
Averill Park, NY). The trap column was prepared in the same
manner but to a length of 1 cm.
The aqueous mobile phase (A) consisted H2O : ACN : FA
(95 : 4.9 : 0.1 v/v/v) and the organic mobile phase (B) of ACN : FA
(99.9 : 0.1 v/v). Column flow was 250 nl/min. Next, 2 ml of antibi-
otic stock solution was injected onto a trap column at 2 ml/min
for 5 min and then eluted onto the column. The column elution
gradient was optimized and was as follows: started from 100%
A (5 min isocratic) to 70% B over 25 min; ramp to 90% B for
1 min and hold for 4 min; ramp back to 100% A for 1 min and
hold for 5 min to re-equilibrate. The mass spectrometer was used
as the detector in all of the SRM experiments.
Mass spectrometry
Tandem mass analysis was performed on a Thermo Scientific
(San Jose, CA) LTQ-Velos Orbitrap mass spectrometer using higher
energy collision dissociation (HCD). Antibiotic samples were
dissolved in H2O : ACN : FA (95:4.9:0.1 v/v/v) at a concentration of
50 mg/ml and infused at a rate of 5 ml/min. The spray voltage was
3 Kv, and the isolation width was 1 Da. Spectra were obtained in
the positive ion mode at 100k resolution.
Selected reaction monitoring was performed on an AB/SCIEX
(Framingham, MA) 4000 QTRAP hybrid triple quadrupole/linear
ion trap mass spectrometer with a nanoflow source. The mass
spectrometer was operated in the positive ion mode for all exper-
iments under the following conditions: curtain gas: 15 psi; colli-
sion gas: high (4 Â 10À5 torr); spray voltage: 2.5 kV; ion source
gas 1: 15 psi; ion source gas 2: 0 psi; interface heater temperature:
70 ꢀC; Q1 and Q3 resolution: low; scan time: 500 ms. The
declustering potential, entrance potential, collision energy, and
collision exit potential were tuned for each individual antibiotic
(5 mg/ml in H2O : ACN : FA, 50 : 49.9 : 0.1 v/v/v) with an infusion
flow of 5 ml/min to obtain the highest sensitivity for each antibi-
otic. The instrument was calibrated by using AB/SCIEX PPG cali-
bration standard and tuned to the manufacturer’s
specifications. Data were acquired and processed with AnalystW
Software, version 1.4.1 (AB/SCIEX), and the data were plotted
for publication by using Sigma Plot, version 11.0 (Systat Software,
Chicago, IL).
Figure 1. (a) Hydrolysis of b-lactam antibiotics by b-lactamase and (b) the
acetylation of chloramphenicol by chloramphenicol acetyltransferase. The
hydrolysis products of b-lactam antibiotics exhibit no antibacterial activity.
Both 3-O-acetylchloramphenicol and 1,3-O,O-diacetylchloramphenicol also
exhibit no antibacterial activity.
Fig. 1(b). Acetylation first occurs at the C-3 hydroxyl moiety. This
acetyl moiety then shifts to the C-1 hydroxyl group via a
nonenzymatic rearrangement. This product is then again acetylated
at the C-3 site to form a diacetylated product. The hydrolyzed
versions of b-lactam antibiotics and the acetylated versions of
chloramphenicol have no antimicrobial activity. Therefore, the
presence or absence of these antibiotics (modified or unmodified)
by SRM can determine whether an organism is expressing the
corresponding antibiotic-inactivating protein (in this case b-lactamase
or chloramphenicol acetyltransferase).
Table 1 lists the SRM transitions for the antibiotics used in this
study. Each transition was optimized by infusing the sample into
the mass spectrometer and adjusting the MS parameters to
obtain the highest sensitivity. Although several unique transitions
for each antibiotic were investigated, we list those that resulted in
high sensitivity, greater signal-to-noise ratio, and no overlap in
transition m/z values among different antibiotics.
3-O-acetylchloramphenicol and 1,3-O,O-diacetylchloramphenicol,
the acetylated products from the interaction of chloramphenicol
with chloramphenicol acetyltransferase-expressing bacteria, were
also investigated to refine this SRM approach. Chloramphenicol
and its acetylated forms were analyzed on an Orbitrap mass
spectrometer to obtain structural and empirical formulas for
the product ions in the MS/MS spectra. For chloramphenicol
Results and discussion
Different antibiotic classes were evaluated in this study. Because
b-lactam antibiotics are one of the most common varieties of
antibiotics, we chose several antibiotics with different susceptibil-
ity to a particular b-lactamase. Ampicillin is an early b-lactam
antibiotic of the penicillin class, whereas piperacillin is a more
recent form that is active against a broader range of bacteria
and more resistant to certain b-lactamases. Nafcillin and
cloxacillin are also of the penicillin class but exhibit an even higher
degree of resistance to certain b-lactamases. Cefoperazone is a
b-lactam antibiotic of the cephalosporin class that is susceptible
to only very limited types of b-lactamase. The hydrolytic action
of b-lactamase on penicillins and cephalosporins is represented
in Fig. 1(a). In the case of b-lactam antibiotics, ring opening
occurs at the nitrogen–carbonyl site via hydrolysis.
Chloramphenicol is a protein synthesis inhibitor susceptible to
acetylation by chloramphenicol acetyltransferase, as illustrated in
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Monitoring bacterial resistance by SRM
Table 1. Table of selected reaction monitoring (SRM) settings
Antibiotic
SRM transition (m/z) Q1 Q3
DPa
EPb
CXPc
CEd
Ampicillin
350.1 160.1
646.1 143.1
436.1 277.0
415.1 199.1
518.2 143.1
323.0 275.0
365.0 275.0
365.0 347.0
60
70
70
70
70
45
45
45
11
10
7
11
10
7
19
50
23
30
30
21
25
22
Cefoperazone
Cloxacillin
Nafcillin
10
8
10
7
Piperacillin
Chloramphenicol
3-O-acetylchloramphenicol
1,3-O,O-diacetylchloramphenicol
8
8
8
8
8
8
aDeclustering potential voltage.
bEntrance potential voltage.
cCollision exit potential voltage.
dCollision energy voltage.
(Fig. 2(a)), m/z 275 and m/z 305 were the major product ions with
the m/z 305 peak resulting from the loss of the C1 hydroxyl
moiety and the m/z 275 peak from the cleavage of the C3
carbon, resulting in the loss of methanol in addition to the
previous C1 hydroxyl loss. The loss of the C1 hydroxyl moiety
was also observed for 3-O-acetylchloramphenicol, which yielded
a product ion at m/z 347 (Fig. 2(b)). In the case of 1,3-O,O-
diacetylchloramphenicol where the hydroxyl group had been
acetylated to form an acetoxy moiety, its loss from the C1 carbon
is instead observed (Fig. 2(c)) and also resulted in the product
ions at m/z 347.
Therefore, we chose the 323 > 275 transition for monitoring
chloramphenicol. For 3-O-acetylchloramphenicol, the transition
365 > 275 was used, as it had better sensitivity and signal-
to-noise after tuning than did the 365 > 347 transition. The
transition 407 > 347 was used for 1,3-O,O-diacetylchloramphenicol.
Other product ions observed in the product ion spectrum did not
yield acceptable sensitivity when used as an SRM transition.
The elution times also varied on the basis of the amount of
acetylation. The addition of acetyl moieties to chloramphenicol
resulted in increased retention time on the column. The
acetylation of the C1 hydroxyl group increased the retention time
from 12 to 14 min. The addition of the second acetyl group on
the C3 hydroxyl group increased it further to 15.5 min. All
three forms of chloramphenicol were, therefore, easily separated
by chromatography.
For chloramphenicol, SRM transitions 323 > 305 and 323 > 275
both had similar sensitivities on the QTRAP after tuning.
However, during the chromatography, the 323 > 305 transition
had a higher background than did the 323 > 275 transition.
Figure 2. Product ion spectrum of (a) chloramphenicol, (b) 3-O-acetylchloramphenicol, and (c) 1,3-O,O-diacetylchloramphenicol. Moieties on the C1 carbon
were found to be the most volatile and lead to cleavage. Loss of the hydroxyl group on the C1 carbon (and subsequent dehydration) is observed for both
chloramphenicol and 3-O-acetylchloramphenicol. The loss of the acetyl group in the C1 position is observed for 1,3-O,O-diacetylchloramphenicol.
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A. M. Haag et al.
All b-lactam antibiotics in this study exhibited good ionization
efficiencies with limits of detection in the low picogram range.
Previous authors have performed SRM for chloramphenicol in
the negative ion mode.[26,27] However, when using the SRM assay
described herein, we found that chloramphenicol, and its
acetylated forms, ionized very efficiently in positive ion mode. A
linear regression of varying concentrations of chloramphenicol
is given in Fig. 3, which illustrates both good linearity and sensi-
tivity of the antibiotic in positive ion mode. In addition, the acet-
ylated forms of chloramphenicol were found to be close to the
same limits of detection and sensitivity of native chlorampheni-
col. Of the b-lactam antibiotics, there was approximately one or-
der of magnitude greater sensitivity for cloxacillin and nafcillin by
SRM than for the other b-lactam antibiotics.
Because ampicilloic acid is the major product from the
hydrolysis of ampicillin with b-lactamase, we wanted to develop
an SRM for its detection. However, we were not able to easily
observe ampicilloic acid by mass analysis, possibly because of
the high volatility of the amine group and its possible degrada-
tion in the ion source. When we performed a parent ion scan
(m/z = 160.1) of hydrolyzed ampicillin, we identified a doubly
charged ion at m/z = 358.7 that directly correlated with the
amount of hydrolyzed ampicillin. We did not investigate the
identity of this ion, but it may be the result of a dimerization of
ampicillin and/or one of its hydrolyzed products, a process
observed by others.[28,29]
cloxacillin and cefoperazone, which are both resistant to the
hydrolyzing effects of this b-lactamase. Chloramphenicol
should also be unaffected by this enzyme because it is not
of the b-lactam class of antibiotics. The ability of this particu-
lar b-lactamase enzyme to hydrolyze the aforementioned
antibiotics is represented in Fig 4. Prior to introducing the enzyme
to a solution comprising the six antibiotics, each antibiotic was
clearly observed by SRM (Fig. 4(a)). After adding the enzyme and
incubating for 2 h, the antibiotics ampicillin, piperacillin, and
nafcillin were completely hydrolyzed and therefore no longer
observed (Fig. 4(b)). However, cloxacillin, cefoperazone, and
chloramphenicol appeared to be unhydrolyzed by the enzyme as
predicted. SRM was therefore able to simultaneously monitor the
susceptibility of a number of different antibiotics to a b-lactamase
in a single analysis.
We also wanted to ascertain whether SRM can determine if an
organism growing in culture is resistant to a particular antibiotic.
For example, by monitoring the concentration of chlorampheni-
col and/or its acetylated forms in the growth media, one can
determine not only if an organism is resistant to chloramphenicol
but also the rate at which the antibiotic is modified. For this
reason, we decided to observe the effects of a chloramphenicol-
resistant strain of E. coli grown in M9 broth-containing chloram-
phenicol. E. coli in this experiment was transformed by the plasmid
pACYC184, which encodes a chloramphenicol resistance gene.[31]
The plasmid allows E. coli to express the enzyme chloramphenicol
We also investigated the ability of SRM to determine the
specificity of a particular b-lactamase enzyme to hydrolyze
the selected group of antibiotics mentioned earlier. The type
of b-lactamase that a bacterium expresses will determine
which b-lactam antibiotic the enzyme is able to hydrolyze.
More recent Food and Drug Administration (FDA)-approved
antibiotics are designed to make them less susceptible to the
actions of b-lactamase. However, because of selective pressure on
the organism by newer and more recently approved antibiotics,
changes in the enzyme allow it to also hydrolyze many, if not all,
of the later classes of antibiotics.
An SRM method was developed that would allow us to deter-
mine the effect of a specific b-lactamase enzyme to a number of
antibiotics. A b-lactamase obtained from the antibiotic-resistant
bacterium Bacillus cereus was chosen for this experiment because
of its well documented specificity to hydrolyze certain b-lactam
antibiotics.[30] Ampicillin, piperacillin, and nafcillin are susceptible
to hydrolysis by this b-lactamase. This is not the case with
Figure 4. Selected reaction monitorings of six different antibiotics
(amp = ampicillin, cef = cefoperazone, chlor = chloramphenicol, clox =
cloxacillin, naf = nafcillin, and pip = piperacillin): (a) before the introduc-
tion of a b-lactamase enzyme from B. cereus and (b) after the reaction
of the enzyme with the antibiotics. All of the antibiotics hydrolyzed by
b-lactamase are known to be susceptible to the enzyme. Cloxacillin and
cefoperazone, both known to be resistant to this specific b-lactamase,
are observed in the spectrum. Chloramphenicol was also unaffected, as
expected, as it is not a b-lactam antibiotic and therefore not hydrolyzed
by b-lactamases.
Figure 3. Linear regression for the detection of chloramphenicol in the
ranges of 2.5–500 ng/ml (12.5 pg to 2.5 ng total chloramphenicol/injec-
tion) using transition 323.0 > 275.0. Coefficient of determination
(R2) = 0.9954.
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Monitoring bacterial resistance by SRM
Figure 5. Selected reaction monitorings of M9 broth containing either chloramphenicol or ampicillin were analyzed at 1-h intervals. (a) At time = 0,
Escherichia coli (pACYC184) was both added to broth-containing chloramphenicol and incubated at 37 ꢀC. Samples were reanalyzed after every hour
to monitor the reduction of chloramphenicol and the formation of both 3-O-acetylchloramphenicol and 1,3-O,O-diacetylchloramphenicol. At 0 h, only
chloramphenicol was observed (rt = ~12 min). However, at 3 h, a significant amount of 3-O-acetylchloramphenicol (rt = ~14 min) was observed, indicat-
ing that the bacteria were resistant to chloramphenicol. At 6 h, almost all chloramphenicol had been acetylated. (b) The same experiment but using b-
lactamase expressing E. coli (pUC18) and ampicillin. After 1 h, all ampicillin was hydrolyzed by the organism. (c) E. coli (C600), which is susceptible to
chloramphenicol, did not result in any change in chloramphenicol concentration. (d) E. coli (C600) also did not result in the hydrolysis of ampicillin
and is therefore susceptible to this antibiotic.
acetyltransferase, which inactivates chloramphenicol via acetylation.
The decrease in available unmodified chloramphenicol and the
increase in the concentration of acetylated forms would indicate
that the organism is resistant to chloramphenicol. This is
illustrated in Fig. 5(a) where the growth media was sampled
every hour and assayed by SRM. At the starting point (time = 0)
when the bacteria were first introduced into the growth media,
only unmodified chloramphenicol (retention time 12 min) was
observed in the growth media. However, after only 3 h of
bacterial growth, a significant amount of 3-O-acetylchloramphenicol
(retention time 14 min) was observed, indicating that the E. coli was
expressing chloramphenicol acetyltransferase and therefore acety-
lating the chloramphenicol. After 6 h, almost no chloramphenicol
was observed, as it had been entirely converted to the acetylated
form. Also at 6 h, there was a significant amount of the diacetylated
chloramphenicol observed (retention time 15.5 min). Because the
analysis time for performing SRM was less than 30 min, the rate-
limiting step for determining chloramphenicol resistance in E. coli
was the time necessary for bacterial growth, a factor beyond our
immediate control.
bacterial growth. At initial conditions, only ampicillin was observed,
but after 1 hr, no ampicillin was observed in the growth media. By
using this method, the time required to determine ampicillin
resistance in E. coli was only 1 h because of the rapid hydrolysis of
ampicillin by the enzyme. As a side note, when ampicillin was
added to a broth that had a 1 mM concentration of the B. cereus
b-lactamase, all ampicillin was hydrolyzed within minutes.
The experiments were also performed using a control group,
consisting of E. coli (C600) that did not have any of the antibiotic
resistance plasmids. Figure 5(c) is the SRM of E. coli (C600) that is
susceptible to chloramphenicol. We did not observe any decrease
in the concentration of chloramphenicol over an 8-h period,
indicating that the E. coli did not express any chloramphenicol
acetyltransferase and was therefore susceptible to chlorampheni-
col. This was also the case when the organism was grown with
ampicillin present, as seen in Fig. 5(d). The concentration of
ampicillin remained constant over the 8-h period, and no hydrolysis
was observed (Fig. 5(d)), thus indicating the susceptibility of E. coli
(C600) to ampicillin.
The same experiment was repeated, but instead E. coli was
transformed with the ampicillin resistance plasmid pUC18, which
allows the organism to express a b-lactamase enzyme that
specifically hydrolyzes ampicillin.[32] Figure 5(b) represents the
SRM assay specific for ampicillin sampled after every hour of
Conclusions
We have shown that SRM is both a rapid and effective technique
for determining the susceptibility or resistance of an organism to
J. Mass Spectrom. 2013, 48, 732–739
Copyright © 2013 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jms

A. M. Haag et al.
antibiotic treatment. The Kirby–Bauer assay remains the gold
standard for determining antibiotic resistance; however, this
technique has drawbacks. It requires one to grow the organism
before antibiotic sensitivity can be determined. It also requires
the isolated organism to be recultured and a zone test
performed. This adds to the time required to perform the assay
and thereby delays effective antibiotic therapy. Therefore,
reducing the time necessary to perform a susceptibility test is
one of the most important benefits to performing the analysis
by SRM.
We have found that SRM can detect modifications of antibiotics
that occur in a short period. In the case of b-lactam antibiotics, the
hydrolysis occurs almost immediately, and therefore, the limiting
factor is the time required to perform the SRM analysis. Current
susceptibility testing can take anywhere from 48 to 72h to perform.
However, SRM analysis can be performed in less than an hour. This
reduces the time necessary to determine the susceptibility of an
organism to any given antibiotic.
a high initial cost. However, this initial cost would eventually
be offset by alleviating the need for the other time-consuming
chemical protocols and outweighed by the multiple advantages
of SRM listed previously. Future work will focus on using this
method with real biological samples and investigating its use
in a clinical setting.
Acknowledgements
The authors wish to thank Robert English and Bo Xu for their
technical expertise and also Richard Hodge and Linda Hackfeld
of the UTMB Organic Synthesis Core facility.
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