ORIGINAL ARTICLE

Insignificant β-lactamase activity of human serum albumin:no panic to nonmicrobial-based drug resistanceM.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) pos-sesses b-lactamase activity, owing to its ability to cleave nitrocefin, and thus contributes to antibioticresistance. Also, its b-lactamase activity is augmented when exposed to pollutants. As nitrocefin is notan 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. Thefindings showed that HSA-mediated hydrolysis of b-lactam antibiotics does not contribute to antibioticresistance.

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: asad.k@rediffmail.com

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 126�7 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 treat-

ment of infections by Gram-negative bacteria (Livermore

1998; Bush 2010). The binding and hydrolysis of b-lactamantibiotics 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

Letters in Applied Microbiology ISSN 0266-8254

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 con-

tribute 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–containingchemical 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, cefo-

taxime, ceftazidime and cefepime were determined in the

absence and presence of environmental pollutants (1-naph-

thol 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 sig-

nificant 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 parame-

ters 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 char-

acteristic of an enzyme. The kinetic parameters (kcat and

Km) deduced from the analysis of Michaelis–Menten plot

(Fig. 1) were 12�8 � 2 s�1 and 98 � 8 lΜ, respectively.

The overall catalytic efficiency (kcat/Km) of HSA on nitr-

ocefin was found to be 1�31 9 105 per mol l�1 s�1

(Table 1). On the other hand, the kinetic parameters (kcatand Km) of recombinant CTX-M-15 on nitrocefin hydro-

lysis were 582�0 � 20 s�1 and 35�0 � 4 lΜ, respectively

(Faheem et al. 2013). The overall catalytic efficiency (kcat/

Km) of recombinant CTX-M-15 on nitrocefin was

1�66 9 107 per mol l�1 s�1 (Faheem et al. 2013). Our

results showed that kcat of HSA-mediated nitrocefin

hydrolysis was 45�5 times lower, while Km was 2�8 times

higher than that of recombinant CTX-M-15. Moreover,

the overall catalytic efficiency (kcat/Km) of HSA on nitr-

ocefin hydrolysis was 126�7-folds lower than that of

recombinant CTX-M-15. Our results strongly suggest that

2·0

1·6

1·2

0·8

0·4

0·00 100 200

[Nitrocefin], µmol [Nitrocefin], µmol300 400 500 0 100 200 300 400 500

V0

× 1

0–4 ,

mol

s–1

2·0

1·6

1·2

0·8

0·4

0·0

V0

× 1

0–4 ,

mol

s–1

(a) (b)

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 l�1) 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 l�1). The vertical bars represent standard deviation (SD) of the mean.

326 Letters in Applied Microbiology 57, 325--329 © 2013 The Society for Applied Microbiology

Insignificant b-lactamase activity of HSA M.T. Rehman et al.

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 cata-

lytic activity of HSA in the presence of such environmen-

tal 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 con-

tribute 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 nitr-

ocefin 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 18�7 � 2 s�1, respectively. On the other

hand, in the presence of 1 : 2 and 1 : 5 (HSA:2-naphthol),

the kcat values were 14�5 � 3 and 17�0 � 4 s�1, 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 77�7 � 6 and 51�9 � 4

lmol l�1, respectively. On the other hand, in the presence of

1 : 2 and 1 : 5 (HSA:2-naphthol), the Km values were

decreased to 83�5 � 7 and 61�5 � 3 lmol l�1, 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 1�93 9 105 and 3�60 9 105 per mol l�1

s�1, respectively. The corresponding values in the presence of

1 : 2 and 1 : 5 (HSA:2-naphthol) were 1�74 9 105 and

2�76 9 105 per mol l�1 s�1, 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 (1�3–2�7 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 moni-

tored the hydrolysis of antibiotics of cephalosporin group

(cefazolin, cefuroxime, cefotaxime, ceftazidime and cefe-

pime) 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 kinet-

ics could not be studied. Instead, we determined the spe-

cific 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 0�79 � 0�03, 0�96 � 0�05, 0�10 � 0�02,0�04 � 0�01 and 0�02 � 0�01 lmol min�1 lg�1, respec-

tively, 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 min�1 lg�1 (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 1 Kinetic parameters of human serum albumin (HSA) on nitr-

ocefin in the absence or presence of different environmental pollu-

tants

Kinetic parameters*

Km (lmol l�1) kcat (s�1)

kcat/Km

(per mol l�1 s�1)

Recombinant

CTX-M-15

35�0 � 4 582�0 � 20 1�66 9 107

HSA alone 98�0 � 8 12�8 � 2 1�31 9 105

HSA + 1-naphthol

(1 : 2)

77�7 � 6 15�0 � 3 1�93 9 105

HSA + 1-naphthol

(1 : 5)

51�9 � 4 18�7 � 2 3�60 9 105

HSA + 2-naphthol

(1 : 2)

83�5 � 7 14�5 � 3 1�74 9 105

HSA + 2-naphthol

(1 : 5)

61�5 � 3 17�0 � 4 2�76 9 105

*The results are represented as mean � SD of three independent

experiments.

Table 2 Specific activity of human serum albumin (HSA) on different antibiotics of cephalosporin group in the absence and presence of different

environmental pollutants

Antibiotic*

Specific activity (lmol min�1 lg�1)†

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)

Cefazolin (I) 1640 � 8 0�79 � 0�03 0�80 � 0�04 0�82 � 0�03 0�81 � 0�04 0�87 � 0�05Cefuroxime (II) 1134 � 6 0�96 � 0�05 0�98 � 0�06 0�99 � 0�04 0�92 � 0�04 0�96 � 0�03Cefotaxime (III) 1029 � 7 0�10 � 0�02 0�09 � 0�02 0�10 � 0�03 0�08 � 0�02 0�11 � 0�03Ceftazidime (III) 903 � 3 0�04 � 0�01 0�04 � 0�01 0�05 � 0�01 0�03 � 0�01 0�04 � 0�01Cefepime (IV) 691 � 4 0�02 � 0�01 0�02 � 0�01 0�03 � 0�01 0�03 � 0�01 0�04 � 0�01

*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

M.T. Rehman et al. Insignificant b-lactamase activity of HSA

(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-naph-

thol) 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 deter-

mined the MICs of different cephalosporin antibiotics after

incubating them with pollutant exposed HSA. It was

expected that when cephalosporin antibiotics were incu-

bated 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 pol-

lutants, and they remain inefficient to hydrolyse b-lactamantibiotics 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 l�1

Tris-HCl buffer, pH 7�4 on Shimadzu UV-Vis spectro-

photometer (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

(30 to 500 lmol l�1) was used and the HSA concentration

was kept constant at 10 lmol l�1, whereas the concentration

of recombinant CTX-M-15 was 0�44 nmol l�1.

v ¼ Vmax ½S�=Km þ ½S� ð1Þkcat ¼ Vmax=½E� 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 cephalo-

sporin group was performed in 20 mmol l�1 Tris-HCl

buffer, pH 7�4 on Shimadzu UV-Vis spectrophotometer

(UV-1800) at 30°C (Francisco et al. 2008). Different anti-

biotics used were nitrocefin (De482 = +15 000 per

mol l�1 cm�1), cefazolin (De320 = +1067 per mol l�1

cm�1), cefuroxime (De262 = �8540 per mol l�1 cm�1),

cefotaxime (De264 = �7250 per mol l�1 cm�1), ceftazi-

dime (De265 = �10 300 per mol l�1 cm�1) and cefepime

(De267 = � 9120 per mol l�1 cm�1).

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 concen-

tration of antibiotic substrate used was 200 lmol l�1, and

the concentrations of HSA and recombinant CTX-M-15

used were 10 lmol l�1 and 2–20 nmol l�1, respectively.

One unit of specific activity of HSA or recombinant

CTX-M-15 is defined as the amount of antibiotic hydro-

lysed 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

Table 3 Minimum inhibitory concentration (lg ml�1) 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) 2 2 2 >1024

Cefuroxime (II) 1 1 1 >1024

Cefotaxime (III) 0�25 0�25 0�25 >1024

Ceftazidime (III) 0�25 0�25 0�25 32

Cefepime (IV) 0�25 0�25 0�25 128

*From Faheem et al. (2013).

328 Letters in Applied Microbiology 57, 325--329 © 2013 The Society for Applied Microbiology

Insignificant b-lactamase activity of HSA M.T. Rehman et al.

(1-naphthol and 2-naphthol), by micro-dilution method

with minor modifications, and the results were inter-

preted according to Clinical Laboratory Standards Insti-

tute (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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