Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
Insignificant β-lactamase activity of human serum albumin:
no panic to nonmicrobial-based drug resistance
M.T. Rehman, M. Faheem and A.U. Khan
Medical Microbiology and Molecular Biology Laboratory, Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, UP, India
Significance and Impact of the Study: Earlier reports showed that human serum albumin (HSA) possesses b-lactamase activity, owing to its ability to cleave nitrocefin, and thus contributes to antibiotic
resistance. Also, its b-lactamase activity is augmented when exposed to pollutants. As nitrocefin is not
an antibiotic of clinical use, the conclusion drawn does not represent a true scenario and is misleading.
Our results showed that HSA is inefficient in cleaving nitrocefin as compared to a true b-lactamase
(CTX-M-15) and is practically inactive on cephalosporin antibiotics even in the presence pollutants. The
findings showed that HSA-mediated hydrolysis of b-lactam antibiotics does not contribute to antibiotic
resistance.
Keywords
beta-lactamases, CTX-M-15, human serum
albumin, multi-drug resistance, nonmicrobial
drug resistance.
Correspondence
Asad U. Khan, Medical Microbiology and
Molecular Biology Laboratory, Interdisciplinary
Biotechnology Unit, Aligarh Muslim University,
Aligarh, UP, 202002-India.
E-mail: [email protected]
2013/0633: received 2 April 2013, revised 29
May 2013 and accepted 7 June 2013
doi:10.1111/lam.12116
Abstract
Recently, it was speculated that human serum albumin (HSA) possesses
b-lactamase activity and could contribute to nonmicrobial-based antibiotic
resistance, owing to its ability to hydrolyse the b-lactam ring of nitrocefin.
Moreover, the putative b-lactamase activity of HSA has been shown to increase
significantly in the presence of environmental pollutants (1-naphthol and
2-naphthol). It was postulated that HSA could also cleave the b-lactam ring of
clinically significant antibiotics. We studied the b-lactamase activity of HSA on
clinically significant antibiotics of cephalosporin group in the presence of
environmental pollutants by determining specific activity, enzyme kinetics and
minimum inhibitory concentrations (MIC). The specific activity of HSA on
various cephalosporins was found to be 1181–34 550 times lower than that
observed for recombinant CTX-M-15 (used as positive control). The catalytic
efficiency (kcat/Km) of HSA on nitrocefin hydrolysis was 1267 times lower than
that of recombinant CTX-M-15, and it has increased only 2- to 3-folds in the
presence of environmental pollutants. Moreover, cephalosporins were not
hydrolysed by HSA under experimental conditions. The MIC data also showed
that HSA is incapable of hydrolysing cephalosporins. The study concludes that
HSA is inefficient to cleave antibiotics of cephalosporin group and hence does
not contribute to nonmicrobial-based antibiotic resistance.
Introduction
Antibiotic resistance is a worldwide health problem with
serious socio-economic implications. The resistance to
b-lactam group of antibiotics in Gram-negative bacteria is
mainly observed due to the production of b-lactamases,
which cleave the amide bond in the b-lactam ring
containing antibiotics (Matagne et al. 1990). The
b-lactam-containing antibiotics account for more than
50% of global antibiotic consumption, and cephalosporins
are a major class of b-lactam antibiotics used for the treatment of infections by Gram-negative bacteria (Livermore
1998; Bush 2010). The binding and hydrolysis of b-lactam
antibiotics has therefore important pharmacokinetic and
pharmacodynamic implications as only free and stable
antibiotics are pharmacologically active and can be
absorbed in the body (Briand et al. 1982; Lin et al. 1987).
Thus, the efficacy of an antibiotic to inhibit the growth
of pathogenic bacteria depends upon its stability and
bioavailability in the plasma.
Letters in Applied Microbiology 57, 325--329 © 2013 The Society for Applied Microbiology
325
Insignificant b-lactamase activity of HSA
M.T. Rehman et al.
Human serum albumin (HSA) is the most abundant
protein of the human plasma (Carter et al. 1994; Peters
1995). In addition to its major role in the transportation
of various molecules throughout the body (Bhattacharya
et al. 2000), it possesses several additional features such
as enolase, esterase and hydrolase activities (Nerli and
Pico 1994; Salvi et al. 1997; Yang et al. 2007). It has been
reported that HSA has b-lactamase activity and can contribute to nonmicrobial-based antibiotic drug resistance
by hydrolysing antibiotics of clinical use. Moreover, the
b-lactamase activity of HSA was expected to further
increase in the presence of environmental pollutants
such as naphthols (Ahmad et al. 2012). However, the
b-lactamase activity of HSA has been evaluated by virtue of
its ability to hydrolyse nitrocefin, a b-lactam ring–containing
chemical substrate (Ahmad et al. 2012). Considering the fact
that nitrocefin has not been used as a human therapeutic,
it is important to evaluate HSA-mediated hydrolysis of
clinically relevant b-lactam antibiotics.
In view of the above background, we have initiated our
study to address whether HSA is capable of hydrolysing the
antibiotics that are frequently used in hospital settings and,
thereby, contribute to nonmicrobial drug resistance. For
this purpose, steady-state kinetics and specific activity of
HSA-mediated hydrolysis of various b-lactam antibiotics
of cephalosporin group such as cefazolin, cefuroxime, cefotaxime, ceftazidime and cefepime were determined in the
absence and presence of environmental pollutants (1-naphthol and 2-naphthol). Minimum inhibitory concentrations
(MICs) were also determined on Escherichia coli DH5a after
incubating cephalosporins with environmental pollutant
exposed HSA. A recombinant CTX-M-15 (an extended
spectrum b-lactamase) was used as a positive control.
Results and discussion
This study was aimed to ascertain whether HSA has a significant b-lactamase activity to pose a threat in the form
of nonmicrobial-based drug resistance as reported earlier
(Ahmad et al. 2012). Hence, steady-state kinetic parameters for HSA-mediated hydrolysis of nitrocefin have been
determined, and the results obtained were compared with
a reference b-lactamase, recombinant CTX-M-15. We
found that HSA followed Michaelis–Menten behaviour
and gives a rectangular hyperbolic curve which is a characteristic of an enzyme. The kinetic parameters (kcat and
Km) deduced from the analysis of Michaelis–Menten plot
(Fig. 1) were 128 2 s1 and 98 8 lΜ, respectively.
The overall catalytic efficiency (kcat/Km) of HSA on nitrocefin was found to be 131 9 105 per mol l1 s1
(Table 1). On the other hand, the kinetic parameters (kcat
and Km) of recombinant CTX-M-15 on nitrocefin hydrolysis were 5820 20 s1 and 350 4 lΜ, respectively
(Faheem et al. 2013). The overall catalytic efficiency (kcat/
Km) of recombinant CTX-M-15 on nitrocefin was
166 9 107 per mol l1 s1 (Faheem et al. 2013). Our
results showed that kcat of HSA-mediated nitrocefin
hydrolysis was 455 times lower, while Km was 28 times
higher than that of recombinant CTX-M-15. Moreover,
the overall catalytic efficiency (kcat/Km) of HSA on nitrocefin hydrolysis was 1267-folds lower than that of
recombinant CTX-M-15. Our results strongly suggest that
2·0
2·0 (a)
(b)
V0 × 10–4, mol s–1
V0 × 10–4, mol s–1
1·6
1·6
1·2
0·8
0·4
0·0
1·2
0·8
0·4
0
100
300
200
[Nitrocefin], µmol
400
500
0·0
0
100
200
300
[Nitrocefin], µmol
400
500
Figure 1 Steady-state kinetics of human serum albumin (HSA) on nitrocefin. The figure shows Michaelis–Menten plots of HSA catalysed nitrocefin
hydrolysis. Panel A shows nitrocefin hydrolysis by HSA alone (–●–), in the presence of 1-naphthol at 1 : 2 (–▲–) and 1 : 5 (–■–) molar ratios.
Panel B shows nitrocefin hydrolysis by HSA alone (–●–), in the presence of 2-naphthol at 1 : 2 (–▲–) and 1 : 5 (–■–) molar ratios. HSA
(10 lmol l1) was preincubated in the absence and presence of 1-naphthol and 2-naphthol (at 1 : 2 and 1 : 5 ratios), and the catalytic activity
was measured on different concentrations of nitrocefin (30–500 lmol l1). The vertical bars represent standard deviation (SD) of the mean.
326
Letters in Applied Microbiology 57, 325--329 © 2013 The Society for Applied Microbiology
M.T. Rehman et al.
Insignificant b-lactamase activity of HSA
Table 1 Kinetic parameters of human serum albumin (HSA) on nitrocefin in the absence or presence of different environmental pollutants
Kinetic parameters*
Recombinant
CTX-M-15
HSA alone
HSA + 1-naphthol
(1 : 2)
HSA + 1-naphthol
(1 : 5)
HSA + 2-naphthol
(1 : 2)
HSA + 2-naphthol
(1 : 5)
Km (lmol l1)
kcat (s1)
kcat/Km
(per mol l1 s1)
350 4
5820 20
166 9 107
980 8
777 6
128 2
150 3
131 9 105
193 9 105
519 4
187 2
360 9 105
835 7
145 3
174 9 105
615 3
170 4
276 9 105
*The results are represented as mean SD of three independent
experiments.
HSA is a poor enzyme in hydrolysing even nitrocefin as
compared to recombinant CTX-M-15.
Some environmental pollutants such as 1-naphthol and
2-naphthol alter the conformation of HSA and, thereby,
affect its catalytic activity (Ahmad et al. 2012). If the catalytic activity of HSA in the presence of such environmental pollutants increases up to a level that it acquires the
ability to cleave cephalosporins or other antibiotics of
clinical significance, then there might be a chance to contribute to nonmicrobial-based antibiotic resistance. We
ascertain this by determining the effect of 1-naphthol and
2-naphthol on the catalytic activity of HSA against nitrocefin and other antibiotics (Table 1). The kcat values of
HSA-mediated nitrocefin hydrolysis in the presence of
1 : 2 and 1 : 5 (HSA:1-naphthol) ratios were increased to
15 3 and 187 2 s1, respectively. On the other
hand, in the presence of 1 : 2 and 1 : 5 (HSA:2-naphthol),
the kcat values were 145 3 and 170 4 s1, respectively
(Table 1). Moreover, the Km values of HSA-mediated
nitrocefin hydrolysis in the presence of 1 : 2 and 1 : 5
(HSA:1-naphthol) were decreased to 777 6 and 519 4
lmol l1, respectively. On the other hand, in the presence of
1 : 2 and 1 : 5 (HSA:2-naphthol), the Km values were
decreased to 835 7 and 615 3 lmol l1, respectively
(Table 1). Further, the overall catalytic efficiency (kcat/Km) of
HSA in the presence of 1 : 2 and 1 : 5 (HSA:1-naphthol)
was improved to 193 9 105 and 360 9 105 per mol l1
s1, respectively. The corresponding values in the presence of
1 : 2 and 1 : 5 (HSA:2-naphthol) were 174 9 105 and
276 9 105 per mol l1 s1, respectively (Table 1). It is
evident that the increase in the catalytic efficiency of HSA, on
nitrocefin, in the presence of environmental pollutants is only
marginal (13–27 times only), which is still around 100-folds
lower than that of recombinant CTX-M-15.
The hydrolysis of nitrocefin by HSA does not represent
actual scenario regarding the ability of HSA to cleave
b-lactam antibiotics of clinical significance. So, we monitored the hydrolysis of antibiotics of cephalosporin group
(cefazolin, cefuroxime, cefotaxime, ceftazidime and cefepime) by HSA in the absence and presence of 1-naphthol
and 2-naphthol. As no detectable hydrolytic activity of
HSA was observed on these antibiotics, steady-state kinetics could not be studied. Instead, we determined the specific activity of HSA against these antibiotics (Table 2).
The specific activity of HSA (in the absence of naphthols)
on cefazolin, cefuroxime, cefotaxime, ceftazidime and
cefepime was 079 003, 096 005, 010 002,
004 001 and 002 001 lmol min1 lg1, respectively, whereas the specific activity of recombinant
CTX-M-15 on the respective b-lactam antibiotics was
1640 8, 1134 6, 1029 7, 903 3 and 691 4
lmol min1 lg1 (Table 2). In other words, the specific
activity of HSA was found to be 2076 (cefazolin), 1181
(cefuroxime), 10290 (cefotaxime), 22575 (ceftazidime)
and 34550 (cefepime) times lower than CTX-M-15
Table 2 Specific activity of human serum albumin (HSA) on different antibiotics of cephalosporin group in the absence and presence of different
environmental pollutants
Specific activity (lmol min1 lg1)†
Antibiotic*
Recombinant
CTX-M-15
Cefazolin (I)
Cefuroxime (II)
Cefotaxime (III)
Ceftazidime (III)
Cefepime (IV)
1640
1134
1029
903
691
8
6
7
3
4
HSA alone
079
096
010
004
002
003
005
002
001
001
HSA + 1-naphthol
(1 : 2)
080
098
009
004
002
004
006
002
001
001
HSA + 1-naphthol
(1 : 5)
082
099
010
005
003
003
004
003
001
001
HSA + 2-naphthol
(1 : 2)
081
092
008
003
003
004
004
002
001
001
HSA + 2-naphthol
(1 : 5)
087
096
011
004
004
005
003
003
001
001
*The generation of cephalosporin antibiotics is given in the parenthesis.
†The results are represented as mean SD of three independent experiments.
Letters in Applied Microbiology 57, 325--329 © 2013 The Society for Applied Microbiology
327
Insignificant b-lactamase activity of HSA
M.T. Rehman et al.
Table 3 Minimum inhibitory concentration (lg ml1) of different antibiotics of cephalosporin group in the presence of human serum albumin
(HSA) exposed to pollutants
Antibiotics
DH5a only*
HSA + 1-naphthol
(1 : 5)
HSA + 2-naphthol
(1 : 5)
Recombinant
CTX-M-15
Cefazolin (I)
Cefuroxime (II)
Cefotaxime (III)
Ceftazidime (III)
Cefepime (IV)
2
1
025
025
025
2
1
025
025
025
2
1
025
025
025
>1024
>1024
>1024
32
128
*From Faheem et al. (2013).
(Table 2). Moreover, the specific activity of HSA in the
presence of 1-naphthol and 2-naphthol did not found to
be significant.
It has been hypothesized that the exposure of HSA to
environmental pollutants (such as 1-naphthol and 2-naphthol) has resulted in the gain of structure, which in turn
improved its catalytic activity (12). If this is true, then the
incubation of b-lactam antibiotics of cephalosporin group
to pollutant exposed HSA would have been resulted in the
cleavage of antibiotics. To check this hypothesis, we determined the MICs of different cephalosporin antibiotics after
incubating them with pollutant exposed HSA. It was
expected that when cephalosporin antibiotics were incubated with pollutant exposed HSA, then they would have
been hydrolysed and the MICs on E. coli DH5a would have
been increased. However, the MIC values obtained in the
present study were similar irrespective of the fact that HSA
has been exposed to environmental pollutants or not
(Table 3). This showed that HSA exposed to environmental
pollutants is still inefficient in cleaving antibiotics of clinical
significance.
The study concludes that HSA possessed negligible
activity to hydrolyse b-lactam antibiotics that are used
clinically. Moreover, the catalytic activity of HSA did not
altered significantly in the presence of environmental pollutants, and they remain inefficient to hydrolyse b-lactam
antibiotics of cephalosporin group. Hence, there should
not be any panic due to HSA-mediated nonmicrobial
antibiotic resistance.
Material and methods
Steady-state kinetics on nitrocefin
The steady-state kinetics of HSA and recombinant
CTX-M-15 on nitrocefin was performed in 20 mmol l1
Tris-HCl buffer, pH 74 on Shimadzu UV-Vis spectrophotometer (UV-1800) at 30°C (Galleni et al. 1994). The
kinetic parameters (kcat and Km) were determined by
Michaelis–Menten equations (Equation 1 and 2) as the
results were analysed as reported earlier (Rehman et al.
2012). In these experiments, a varied nitrocefin concentration
328
(30 to 500 lmol l1) was used and the HSA concentration
was kept constant at 10 lmol l1, whereas the concentration
of recombinant CTX-M-15 was 044 nmol l1.
v ¼ Vmax ½S=Km þ ½S
kcat ¼ Vmax =½E
ð1Þ
ð2Þ
where v and Vmax are the initial and maximum velocity
of hydrolysis, respectively; [S] is the concentration of the
substrate used; [E] is the enzyme concentration in the
reaction; Km is the Michaelis–Menten constant; and kcat is
the catalytic activity of the enzyme. The results presented
in this study are the mean SD of three independent
experiments.
Specific activity on b-lactam antibiotics
The hydrolysis of various b-lactam antibiotics of cephalosporin group was performed in 20 mmol l1 Tris-HCl
buffer, pH 74 on Shimadzu UV-Vis spectrophotometer
(UV-1800) at 30°C (Francisco et al. 2008). Different antibiotics used were nitrocefin (De482 = +15 000 per
mol l1 cm1), cefazolin (De320 = +1067 per mol l1
cm1), cefuroxime (De262 = 8540 per mol l1 cm1),
cefotaxime (De264 = 7250 per mol l1 cm1), ceftazidime (De265 = 10 300 per mol l1 cm1) and cefepime
(De267 = 9120 per mol l1 cm1).
The specific activity was determined by measuring the
absorbance at a specific wavelength, characteristic of the
antibiotic used, after 1h incubation at 30°C. The concentration of antibiotic substrate used was 200 lmol l1, and
the concentrations of HSA and recombinant CTX-M-15
used were 10 lmol l1 and 2–20 nmol l1, respectively.
One unit of specific activity of HSA or recombinant
CTX-M-15 is defined as the amount of antibiotic hydrolysed per minute per lg of the enzyme (Francisco et al.
2008).
Minimum inhibitory concentration determination
The MIC for various antibiotics was determined, in the
absence and presence of environmental pollutants
Letters in Applied Microbiology 57, 325--329 © 2013 The Society for Applied Microbiology
M.T. Rehman et al.
(1-naphthol and 2-naphthol), by micro-dilution method
with minor modifications, and the results were interpreted according to Clinical Laboratory Standards Institute (CLSI) guidelines (CLSI 2011). Briefly, HSA was
preincubated with different concentrations of 1-naphthol
and 2-naphthol, and the MICs were determined on E. coli
DH5a cells treated with different concentrations of the
antibiotics (after incubation for 1 h with HSA that has
been exposed to 1-naphthol and 2-naphthol). The MIC
was taken as the lowest concentration that totally inhibits
visible bacterial growth.
Acknowledgements
This work was supported by DBT grants, BT/PR11610/
BRB/10/669/2008 and BT/PR11453/BID/07/296/2009 to
AUK. MTR and MF are thankful to University Grants
Commission for Dr D S Kothari Postdoctoral Fellowship
and Department of Biotechnology for SRF, respectively.
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