Download CHARACTERIZATION OF B-LACTAMASES FROM TWO B

CHARACTERIZATION OF B-LACTAMASES FROM TWO BLACTAM RESISTANT ISOLATES OF PSEUDOMONAS
AERUGINOSA
BY
E.Y. Tohamy a ; M.F. Ghaly a ; M.S. Abel-Sabourb and A.M.A.Zaida
FROM
a b Dept.of Botany, Faculty of Science. Zagazig University, Microbiology Dept
of Genetics, Faculty of Agriculture , Moshthor, Benha branch, Zagazig
University, Egypt.
ABSTRACT
(3-lactamase extracted from two highly (3-lactam resistant isolates of
Pseudomonas aeruginosa was purified and characterized. The results of the
purification steps indicate that the /3-lactamase from P. aeruginosa Z 90 was
purified at 44.6 fold, the specific activity reached to 18.28 units/mgprotein and the
yield percentage was reached 50.15 %, while the (3-lactamase from P.aeruginosa
Z 33 was purified at 42.4 fold, the specific activity reached to 20.8 units/mg
protein and the yield percentage was reached to 61.66 %. With respect to its
optimum pH, the optimum values for (3-lactamase from P.aeruginosa Z 33 and
P.aeruginosa Z 90 were 7.5 and 8.0, respectively. The optimum temperature
was around 40°C for both enzymes from the two isolates. Studying the heat
stability of (3-lactamase revealed that the enzyme of P.aeruginosa Z90 was more
stable than the enzyme from P.aeruginosa Z 33. The enzyme activity was increased
linearly with increasing (3-lactamase concentration with small deviation of
linearity at higher concentrations in the two isolates. Amino acid analysis of the
purified (3-lactamase from the two isolates indicate that alanine was the most
abundant amino acid in the two isolates. Isolucine was the least detected amino
acid in (3-lactamase from P.aeruginosa Z 90 with a percent of 3.12 % while
tyrosine was the least one in P. aeruginosa Z 33 with a per cent of 1.93 %. The
results of inhibition profiles show that (3-lactamase from the two isolates was
strongly inhibited by 0.1 mM CuSO^ but not by EDTA. (3-lactam
inhibitors results indicated that carbenicillin was the stronger inhibitor for the
purified (3-lactamase from P.aeruginosa Z 33, while amoxycillin was the stronger
one for the enzyme from P.aeruginosa Z 90.
INTRODUCTION
p-lactamase has been considered one of the biochemical mechanisms of resistance
to p-lactam antibiotics in bacteria (Sotto et al., 1996). The enzymes from Grampositive bacteria are inducible, extra cellular and have predominantly penicillinase
activity but the enzymes from Gram- negative bacteria are more varied in nature,
but may be classified into several groups by substrate profile, inhibitory profile
and physicochemical and immunological properties (Sato et al., 1982 and Bush et
al., 1991).
Az. J. Microbiol Vol. 53, July, 2001.
Pseudomonas aeruginosa is, an opportunistic human pathogen, responsible for
several infections mainly in immunocompromised hosts (Grimwood, 1992; Bert
and Lambert-Zechovsky, 1996 and Hostacka & Majtan, 1997). These bacteria
cause several infections which are associated with urinary, respiratory, burn
wounds, eyes, ear, as well as nosocomial infections (Ashour, 1980; Kadry, 1985
Hoiby et a/.,1987; Tummler et al., 1991 and Ravaoarinoro et a!., 1996).
The emergence of resistance to the antibiotics specially aminoglycoside and |3lactam antibiotics is a major problem in treating infections with P.aeruginosa
(Radberg et a/., 1990). Bacterial resistance to P-lactam antibiotics is often
associated with the production of p-lactamases which hydrolyze the cyclic amide
bond in the (3-lactam ring of penicillins, cephalosporins and related compounds
(Abraham and Chain, 1940; Bush et al., 1991; Mascaretti et al., 1999 and Yan et
al., 2000). This study aimed to purify and characterize the properties of Plactamase from two highly resistant isolates of clinical P.aeruginosa.
MA TERIALS AND METHODS Tested P.aeruginosa isolates: ,
Two P-lactam resistant isolates of Pseudomonas aeruginosa were isolated from
documented infections at Zagazig University Hospitals, Egypt as described
previously (Ghaly et al., 2001). These isolates were identified by both the
traditional diagnostic biochemical tests according to Wong et al., 1984; Degtyar et
al., 1985 and Gilardi, 1971 and 1992 as well as with the API 2oE system.
These two isolates were designated as P.aeruginosa Z 33 and P.aeruginosa Z 90.
The two tested P.aeruginosa isolates were described for their antibiotics
susceptibility by the standard agar disc diffusion test according to Bauer et al.
(1966) using Mueller-Hinton agar plates (Difco). The results were interpreted
according to the National Committee for Clinical Laboratory Standards (NCCLS,
1999), guidelines were followed wherever possible.
P.aeruginosa Z 33 and Z 90 tested in the present study were multiple resistant to
at least 8 antibiotics namely; ampicillin, amoxicillin, amoxycillin/clavulanic acid
(2:1), cephalexin. cephaloridine, cloxacillin, ceftraiaxone, and cefotaxime (Ghaly
et al., 2001).
Preparation of p-lactamase extract (as described by Pagani et al., 1990):
Two highly P-lactam resistant isolates of P. aeruginosa were grown overnight in
brain heart infusion broth (Difco), then diluted 10-fold with the
same medium and cultured with shaking at 37 C for 5 h. After 5h of incubation,
the bacterial cells were harvested by centrifugation at 4 C, washed twice with
50mM phosphate buffer [pH: 7.0] and suspended in 4 ml of the same buffer. The
cell suspension was sonicated in ultrasonic disrupter (UR-150 P; Tomy Seiko Co.,
Ltd) at 75 W for 3 min. in an ice bath. The disrupted cell suspension was
centrifuged at 10000 ipm for 15 min. at 4 C, the resulting supernatant was used as
a crude enzyme extract.
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Az.J. Microbiol. Vol. 53, July, 2001.
Purification of p-lactamase (according to Cuchural et a/., 1986):
The crude enzyme solution was subjected to ammonium sulphate precipitation
(50-90%). The solutions were precipitated by centrifugation at 10000 rpm, then
each fraction precipitate was dissolved in 5 ml of 0.1 M phosphate buffer [pH:
7.0]. p-lactamase activity was measured and the protein concentration were
determined in each fraction according to Lowrey et al., 1951 with bovine serum
albumin as standard.The active protein fractions were applied to a sephadex G-100
(Sigma Co.) gel filtration column equilibrated with 0.01M potassium phosphate
buffer [pH 8.0] and eluted with the same buffer . Active fractions were applied to
a DEAE -DE 52 -cellulose anion exchange column equilibrated with 0.01 M
potassium phosphate buffer [pH 8.0] and eluted with 0.05 M NaCl in 0.01M
phosphate buffer. All purification steps were performed at
4°C.
P -lactamase assay:
p-Lactamase activity of the crude enzyme and the active fractions in different
steps was assayed spectrophotometrically with nitrocefin according to
O' Callaghan et a/.(1972) and Neu (1989). One unit of p-lactamase activity was
defined as that amount of enzyme that can destroy 1 (i mol of nitrocefin per min.
P-lactamase specific activity of a sample was calculated by dividing the Plactamase activity by the amount of protein (units per milligram) in the reaction
mixture.
B- lactamase activities from the two tested P.aeruginosa isolates were tested for
their optimum pH, optimum temperature, heat stability, substrate concentration
and p-lactamase concentration, according to O,Callaghan et al., 1972 and
Cuchural et al., 1986.
Amino acid analysis:
Amino acid analysis was performed on reverse phase-high pressure liquid
chromatography (Shimadzu LC-10AD, Shimadzu Corporation, Japan) following
hydrolysis of the sample with 6N HC1 at 110°C for 24 h according to (Association
of Official Analytical Chemists AOAC (1995). Samples were analyzed on
Shimpack amino-Na type column (10 x 6 mm) obtained from Shimadzu
Corporation. The post column samples were derivated with O-phthaldialdehyde
(OPA) according to Ishida et al. (1981) and data were integrated using an
integrator model CR7A (Shimadzu chromatopac data processor).
Influences of some non (Mactam and p-Iactam compound on B-Iactamases
activities:
The purified p lactamases from the two isolates of P.aeruginosa (Z 33 and Z 90)
were preincubated with the following compounds for 5 min before the addition of
nitrocefin, the non B-lactam compounds were the following: EDTA and iodine
with a concentration of (lOmM), Cuso4, CoSO4, andFeS04 with a concentration
of 0.1 mMand MgSO4, mNso4 and CaSO4 with a concentration of IMm. The PIactam compounds used were the following p-lactam antibiotics: penicillin,
carbenicillin, cefotaxim, oxacillin and amoxycillin with a concentration of (0.1
Mm). After the incubation period p-lactamase activities were measured.
Al. J. MicrobioL Vol. 53, July, 2001.
RESULTS AND DISCUSSION
The susceptibility of the two studied isolates of P. aeruginosa against p-lactam
antibiotics were summarized in Table 1. The data revealed that P. aeruginosa Z 90
was resistant to the action of all tested antibiotics with the exception of imipenem,
whereas P. aeruginosa Z 33 was resistant to the action of all tested antibiotics with
the exception of imipenem and ceftazidime. These results were in agreement with
that of Williams (1985), where he reported that imipenem has a high potency
against a broad spectrum of microorganisms including P. aeruginosa.
Production of P-lactamase is a major factor in the emergence of resistance during
antimicrobial therapy and associated relapses of infection (Letendre and Turgeon,
1989). Production of P-lactamase from bacteria was reported by many
investigators (Cuchural et al., 1986; Fujii et a/., 1986 Edwards & Green wood,
1990 and Sotto et al., 1996).
The results showed in Fig. 1 (a,b) revealed that 80% saturation of ammonium
sulphate gave maximal value of protein and p-lactamase activity in the two studied
P.aeruginosa isolates (Z 33 and Z 90), where the maximum P-lactamase activities
were 5.8 and 2.9 u mol/min/ml for the two isolates, respectively. This result was in
agreement with the result of Cruchural et al., 1986. The results of the purification
steps (Table 2, Fig 2(a.b) and Fig. 3(a,b) indicate that P-lactamase from
P.aeruginosa Z 90 was purified at 44.6 fold and the specific activity reached to
18.28 units/mg protein and the yield percentage was reached to 50.15 % while the
p-lactamase from P. aeruginosa Z 33 was purified at 42.4 fold and the specific
activity was reached to 20.8 and the yield percentage was reached to 61.66%. The
extremely low specific activities of enzymes from more resistant isolates may
remain a problem ( Britz and Wilkinson, 1978).
In the present investigation , the results (Fig.4) revealed that the optimal pH of Plactamase varied with the strains. The values were 7.5 and 8.0 for P-lactamase
from P.aeruginosa Z 90 and P.aeruginosa Z 33, respectively. This result nearly
coincided with the result of Furth, 1975, where he reported that pH:8 was the
optimum for P-lactamase from P. aeruginosa strain Dalgleish. The small diversity
of optimum pH values may be related to strain of P. aeruginosa used or more
likely to the substrate and/or the buffer used in detecting the activity.
The effect of temperature on p-lactamase activity is shown in Fig.5. The
results show that the optimum temperature was around 40°C for the two
P.aeruginosa isolates. In this experiment, P-lactamase activity was assayed at the
optimum pH for each isolates. The optimum temperature recorded herein is quite
similar to that recorded by Saino et al. (1982).
An experiment was designed to study the heat stability of the purified Plactamase from the two studied P. aeruginosa isolates at 60 C. The results (Fig.6)
show that P-lactamase of P. aeruginosa Z 90 was more stable than that of
P.aeruginosa Z 33.
Studying the effect of enzyme concentration on p-lactamase activity (Fig.7),
resulted in a linear relationship but the straight line deviated from linearity at the
higher concentrations. Also the p-lactamase activities from the two highly resistant
isolates of P. aeruginosa were measured at different concentrations of nitrocefm,
starts from 5ul (2.5 ug) up to 50 ul (25 ug). The results obtained
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Az. J. Microbiol. Vol. 53, July, 2001.
(Fig.8) show that, at lower concentrations, the rate of P-lactamase reaction resulted
in a curve with a sort of linearity. As the concentration of substrate increased, this
linearity decreased until any further increase in the substrate concentration did not
show significant increase or expressed a little decrease in the velocity of the
enzymatic reaction. Indeed, the present results are in accordance with the typical
enzymatic reaction given earlier by Michaelis and Menten(1913).
Amino acid composition of the purified P-lactamase was determined from the two
studied P.aeruginosa isolates. The results shown in Table 3 revealed that fourteen
amino acids were detected with alanine being the most abundant amino acid in the
two isolates, similar results were reported by Kesado etal. (1989). Isolucine was
the least detected amino acid in P.aeruginosa Z 90 with a percent of 3.12 %
whereas tyrosine was the least one in P.aeruginosa Z 33 with a percent of 1.93%.
For evaluating the role of metal ions and chelating agent (EDTA) on the Plactamase activity, eight compounds were studied namely; EDTA, iodine (10
mM), CuSO4, CoSO4, FeSO4 (0.1 mM) and Mg SO4, Mn SO4, Ca SO4 (ImM).
The purified p-lactamase from the two studied isolates was pre incubated with a
test compound in 50 mM phosphate buffer at the optimum temperature and pH
values for each isolates. After the incubation period (20 min.), the remaining Plactamase activity was assayed spectrophotometrically at 482 nm with nitrocefin
as substrate. The data present in Table 4 revealed that the enzyme from the two
studied isolates (Z90 and Z 33) was inhibited by CuSO4, CoSO4 and iodine but
not inhibited by EDTA. The results of this investigation were in accordance with
that of Saino et al. (1982) where they reported that the P-lactamase activities from
Pseudomonas maltophlia were inhibited by iodine and CuSO4. On the other
hand, the inhibitory effect of 10 mM Cor+ which observed in this investigation
was in disagreement with the result of Fujii et al. (1986) on the P-lactamase from
Legionella gormanii. Generally, the inhibition profiles of P-lactamase differs
with the source of the enzyme. Fe^+ caused activation for the P-lactamase
extracted from P. aeruginosa Z33 more than P.aeruginosa Z 90. Mg^+, Mn^+ and
Ca^+ stimulated the P-lactamase activities from the two P.aeruginosa
isolates with the exception of Mg2+ which not stimulate the enzyme from
P.aeruginosa Z 90. Britz and Wilkinson (1978) reported that the addition of ImM
CaC^, MnC^ or MgSO4 did not alter the activity of P-lactamase from
Bacteroides fragilis.
The effect of p-lactam compounds on the purified p-lactamase from the two
P.aeruginosa isolates (Z33, Z90) are shown in Table 5. Five p-lactam inhibitors
with concentration of O.lmM were used namely; penicillin G, carbenicillin,
cefotaxim, oxacillin and amoxycillin. The results are expressed as percentage of
control. The inhibitors could be arranged regarding to their effect on the enzyme in
the following sequence as carbenicillin > penicillin G > amoxycillin > oxacillin >
cefotaxim for the enzyme from P. aeruginosa Z33. On the other hand, they were
as; amoxycillin > oxacillin > carbenicillin > cefotaxim > penicillin G for the
enzyme from P. aeruginosa Z90, these results confirm the results of Britz and
wilkinson (1978).
Az. J. Microbiol. Vol. 53, July, 2001.
113
Table (1): Antibiotic susceptibility of the two clinical P.aeruginosa isolates (Z
33 and Z 90).
Antibiotic*
Potency
P.aeruginosa Z33
P. aeruginosa Z 90
Ampicillin
10 ug
R
R
Amoxycillin
20 ug
R
R
Amoxycillin/clavulanic acid (2:1)
30 ug
R
R
Cephalexin
30 ug
R
R
Cephaloridine
30 ug
R
R
Cloxacilin
5ug
R
R
Ceftriaxone
30 ug
R
R
Ceftazidime
30 ug
S
R
Cefotaxim
30 ug
R
R
Imipenem
10 ug
s
s
*AI1 antibiotic discs were purchased from Oxoid Co., England except methicillin which was
purchased from BioMerieux Co.,France.
R : Resistant S : Susceptible
Table (2): Purification steps of p-Iactamases from P. aeruginosa Z 90 and P.
aeruginosa Z 33.
Activity
Protein
Specific
Purification
yield %
Purification steps
(umol/min/ml)
(mg/ml)
activity
(units/nig)
fold
6.56
14.69
0.41
1
100
Ammonium sulphate 5.63
10.57
0.53
1.29
85.82
SephadexGlOO
4.12
3.63
1.13
2.76
62.80
DEAE Cellulose
3.29
0.180
18.28
44.59
50.15
Crude extract
5.06
10.30
0.49
1
100
Ammonium sulphate
4.35
7.50
0.58
1.18
85.97
SephadexGlOO
4.09
2.43
1.68
3.43
80.83
DEAE Cellulose
3.12
0.15
20.8
42.4
61.66
aeruginosa Z 90
Crude extract
icruginosa Z 33
Az. J. Microbiol. Vol. 53, July, 2001.
Table (3): Amino acid composition of p-lactamase (mixed fractions) from two
p-lactam resistant isolates of P.aeruginosa (Z 90 and Z 33).
Amino acid
The total detected amino acids ( % )
P.aeruginosa Z 90
P.aeruginosa Z 33
Aspartic acid
10.38
11.10
Threonine
5.40
6.20
Serine
5.87
6.73
Glutamic acid
11.73
12.52
Glycine
9.93
11.48
Alanine
13.09
14.44
Valine
4.84
6.15
Isoleucine
3.12
3.45
Leucine
9.26
8.42
Tyrosine
3.66
1.93
Phenylalanine
3.64
2.66
Histidine
6.87
2.94
Lysine
6.26
6.39
Arginine
5.95
5.59
Table (4): Influences of non- p-lactam compounds on the activity of Plactamase from P.aeruginosa Z 90 and Z 33.
Non p-lactam compound
P-lactamase activity as % of control
P.aeruginosa Z 90
P.aeruginosa Z 33
EDTA(lOmM)
101.19
101.02
Iodine (lOmM)
96.40
94.81
CuSO4(0.1mM)
58.86
60.63
CoSO4(0.1mM)
92.19
92.72
FeS04(0.1mM)
104.20
129.52
M
92.67
124.66
198.92
316.89
§ S04 (ImM)
Mn S04 (lmM)
Ca SO4 (imM)
256.81
352.06
Table (5): Influences of p-lactam compounds on the activity of the purified Plactamase from P.aeruginosa Z 90 and Z 33.
p-lactam compound (O.lmM)
P-lactamase activity (as % of control)
P. aeruginosa Z 90
Penicillin
G
Carbenicillin 95.665 28.259 36.684
Cefotaxim Oxacillin Amoxycillin
27.452 18.479
P. aeruginosa Z 33
35.165 32.454 43.829
39.739 38.771
Az. J. Microbiol. VoL 53, July, 2001. 121
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