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Detection of
metallo-β
-lactamase in
Pseudomonas
aeruginosa
from Tripoli, Libya

Vol. 2 No. 1:3
doi: 10.3823/1014

Mohamed S Ellabib1, Mohamed A Aboshkiwa1, , Nagwa
A Almargani, 2 ; Abdulaziz A. Zorgani1, , Allaaeddin El
Salabi3,4 and Mohammed El-Gumati1
1 Department of Medical
Microbiology and Immunology,
Faculty of Medicine, Al-Fateh
University, Tripoli, Libya,
2 Academy of Graduate Science,
Tripoli, Libya,

3 Department of Infection,
Immunity & Biochemistry,
School of Medicine, University
Hospital of Wales, Heath
Park, Heath, Cardiff, CF14
4XN, UK,
4  Department of Environmental
Health, Faculty of Public
Health, Medical Campus,
University of Benghazi,
Benghazi, Libya.

Correspondence:

sallbros@yahoo.ca

Abstract
Metallo-beta-lactamase (MBL)-producing Pseudomonas aeruginosa isolates have
been reported to be an important cause of nosocomial infections. As very little
information of the antibiotic resistance in Libya is known, this work was undertaken. The present study was conducted to study the incidence of MBLs in P.
aeruginosa isolated from patients admitted to Tripoli Medical Center (TMC) and
to Burn and plastic Surgery Hospital (BPSH). Three hundred and twelve isolates of
P. aeruginosa were cultured from different samples from patients. Isolates were
identified using Conventual’s method and API20 NE. The isolates were subjected
to susceptibility testing using CLSI guidelines. They were further screened for the
production of MBLs by disc potentiating testing using EDTA-impregnated imipenem discs. Of the total 312 isolates of P. aeruginosa, antimicrobial susceptibly tests
to fifteen anti-pseudomonal antibiotic showed that the percent of resistance was
as follows; Amikacin (23.1%), Aztreonam (22.8%), Carbenicillin (51.9%), Cefepime
(28.2%), Cefotaxime (51%), Ceftazidime (29.5%), Ciprofloxacin (26.9%), Gentamicin (35.3%), Imipenem (13.8%), Meropenem (15.7%), Piperacillin (39.1%), Piperacillin/tazobactam (27.6%), Polymyxin B (13.8%), Ticarcillin (53.8%), Tobromycin (31.1).
Thirty four of imipenem resistant isolates were found to be MBL positive (10.9%).
The study showed that MBL screening tests are simple and easy to perform as
routine diagnostic tests for the detection of MBL production. EDTA disk screening
test is simple to perform and to interpret and can easily be introduced into the
workflow of a clinical laboratory. We recommend that all IPM non susceptible P.
aeruginosa isolates be routinely screened for MBL production using the EDTA disk
screen test and that PCR confirmation be performed at a regional laboratory.

This article is available from:
www.jbiomeds.com

Keywords:  Carbapenem resistance, metallo-β-lactamase, Pseudomonas aeruginosa, Tripoli, Libya.

Introduction
The introduction of carbapenems into clinical practice represented a great advance for the treatment of serious bacterial
infections caused by beta-lactamase resistant bacteria [1]. This
is due to the broad spectrum activities and stability to hydrolysis by most beta-lactamase enzymes, which have made carbapenems the drug of choice for the treatment of infections
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caused by penicillin or cephalosporin-resistant Gram-negative
bacilli, particularly extended spectrum beta-lactamase (ESBL)
[2]. However in recent year’s emergence of carbapenem-hydrolysing metallo-beta-lactamase (MBL) in P aeruginosa has
been described worldwide [3]. With respect to infection with
MBL-producing strains represents a therapeutic problem due
to their resistance to all beta-lactams except monobactams
[4]. Several types of MBL enzymes have been identified and

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described in P. aeruginosa such as IMP, VIM. [2, 5]. P. aeruginosa is uniquely problematic due to it is ability to inherent and
acquire resistance to many drug classes via mutation to all
relevant treatments [4]. Furthermore, few isolates of P. aeruginosa are resistant to all reliable antibiotics and this problem
seems likely to grow with the emergence of class 1 integrons
encoding both carbapenems and amikacin resistance genes
[6].The rates of the occurrence of metallo-β-lactamase mediated resistance in P. aeruginosa have escalated since 2000
and severely affected treatment options in Asia, Europe and
Latin America to non-β-lactam antibiotics [8]. Clinical isolates
harboring metallo-β-lactamases have also recently been reported in western Canada and in Texas, which highlight the
need for development of accurate diagnostic tests by clinical
laboratories to detect the presence of new, and more potent
antimicrobial agents [9]. In this respect nosocomial infections
caused by P. aeruginosa remains one of the most difficult to
treat and to control [9]. In vitro antimicrobial susceptibility
data are required for successful therapy, because acquired resistance to such antimicrobials as β-lactams, fluoroquinolones
and aminoglycosides is so prevalent in P. aeruginosa [10-11].
Strategies for controlling P. aeruginosa infections include;
early detection of P. aeruginosa as the causative pathogen,
determination of its antimicrobial susceptibilities, initiation
of effective and adequate therapy as well as strict infection
control practice such as hand hygiene [11].
Up to our knowledge these information and such studies in
Libya are not well known. For that reason this work aims to
obtain some insight view on the detection and prevalence
of such important type of antibiotic resistance mechanisms
in P. aeruginosa in two hospitals; one major teaching Tripoli
Medical Center (TMC) and the other is an important setting
hospital; Burn and Plastic Surgery Hospital (BPSH).

Vol. 2 No. 1:3
doi: 10.3823/1014

and Laboratory Standard Institute recommendations (CLSI)
[13]. The following anti-pseudomonal antibiotics used were
aminoglycosides [amikacin (30µg), gentamicin (10µg), and
tobramycin (10µg)], cephalosporin’s [cefepime (30µg), ceftazidime (30µg) and cefotaxone (30µg)], fluoroquinolones
[ciprofloxacin (5µg)], carbapenems [imipenem (10µg) and meropenem (10µg)] ,carbenicillin (100µg) ticacillin (75µg), pipercillin (100µg), piperacillin/tazobactam (100/10µg), aztreonam
(30µg) and polymyxin B (100µg). Isolates were considered to
be carbapenem sensitive when the zone size around imipenem was ≤13 mm, intermediate 14-15 mm and resistant to ≥16
mm. MBL-producing P. aeruginosa isolates were suspected
when the isolate was resistant to imipenem. Screening and
confirmation for the detection of MBL was carried out by
disc potentiating test with EDTA-impregnated imipenem discs
[14, 15, 16].

Metallo-β-lactamase screening test
All P. aeruginosa isolates which showed resistance to imipenem were submitted for further examination for the detection of MBLs production using EDTA (750 µg) combined
disk. The IMP-EDTA combined disk test was performed as described by [16] Test organisms were inoculated onto plates of
Mueller-Hinton agar and optical density was adjusted to 0.5
McFarland standards. A 0.5M EDTA solution was prepared by
dissolving 186.1 g of disodium EDTA 2H 2 O in 1000 ml of
distilled water and adjusting it to pH 8.0 by using NaOH and
the mixture was sterilized by autoclaving. Two 10µg imipenem discs were placed on the plate and 5µl (750µg) of EDTA
solution was added to one of the discs. The inhibition zones
of the imipenem and imipenem-EDTA discs were compared
after 16-18 h of incubation at 35°C. An increase in the zone
size of at least 7 mm around the imipenem-EDTA was recorded as an MBL-positive isolate.

Materials and Methods

Detection of Metallo-β- lactamase by EDTA

Over the period of this study from August 2008 to august
2009, 312 non duplicate isolates of P. aeruginosa were
obtained and isolated from different clinical samples collected from patients admitted to TMC and BPSH hospitals.
Samples were transported and processed in the microbiology laboratory without delay. Isolates were identified by
standard laboratory techniques using conventional method
and Identification was confirmed with the API 20NE system
(BioMe’rieux, Marcy l’E’toile, France) as well as oxidase test
[12]. Antimicrobial sensitivity testing was performed on Mueller Hinton Agar plates with commercially available discs obtained from (Oxoid) using Kirby Bauer Disc Diffusion method.
The results were recorded and interpreted following Clinical

Differences between imipenem discs and imipenem/EDTA
discs were regarded positive if the P value was observed <
0.05. Statistically significant difference was found in the resistance pattern of MBL positive and negative isolates for
Imipenem depended on (P <0.05),. Increased zone inhibition
to ≥ 7mm was considered as MBL positive. These results were
clearly discriminated using combined disc cut off ≥ 7mm increase in inhibition zone by using imipenem-EDTA statistical
methods to prove the change in inhibitory zone before and
after using EDTA. P value of less than < 0.05 indicates statistical significance results for this change. Statistical analysis
was performed by linear regression with the help of SPSS 17
.0 statistic software (SPSS Inc).

2

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Results

Vol. 2 No. 1:3
doi: 10.3823/1014

Table 2. Prevalence of metallo β-lactamase in TMC & BPSH.

During the period of this study, a total of 312 non duplicate
isolates of P. aeruginosa isolates were isolated; 170 isolates
(54.5%) were from patients admitted to TMCH and 142
(45.5%) were from patients admitted to BPSH. Specimens
site source from TMC were mostly obtained from urine; 50
(92.6%), while most specimens from BPSH were obtained
from wound swab; 86 (74.8%). Susceptibility pattern showed
that the most effective antibiotics with less frequency of resistance against P. aeruginosa isolates were found to be polymyxin B followed by imipenem and meropenem. The less
active antibiotics were ticarcillin, carbenicillin and cefotaxime.
Table 1 represents the frequency of resistant P. aeruginosa
to 15 anti-pseudomonal antibiotics. Considering carbapenem
resistance during this study, it was found that out of 312
isolates of P. aeruginosa, 42 (13.5%) were found resistant
to imipenem and out of this 34 (10.9%) were found to be
MBL producers as confirmed by the disc potentiating method.
Frequency of MBLS in both hospitals are presented in table
2. Resistance pattern of MBL positive and negative isolates
against antibiotics are presented in table 3. The results clearly
showed that the resistance pattern was higher for both MBLs
positive and negative isolates toward other drugs (Table 3).
Table 1. A 
ntibiotic sensitivity pattern of 312 P. aeruginosa
strains to most common used anti-pseudomonas
antibiotics.
% of resistant

Antibiotics Potency (µg)

53.8

Ticarcillin (75)

51.9

Carbenicillin (100)

51

Cefotaxime (30)

39.1

Pipercilline (100)

35.3

Gentamicin (200)

31.1

Tobromycin 30

29.5

Ceftazidime (30)

28.2

Cefepime (30)

27.6

Piperacillin/tazobactam (100)

26.9

Ciprofloxacin (15)

23.1

Amikacin (30)

22.8

Aztreonam (30)

16.4

Meropenem (10)

13.8

Polymyxin B (300)

13.5

Imipenem (10)

N=312.

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Antimicrobial
IMP/EDTA resistant
10.8%

HOSPTIAL
TMC+BPSH

7.6%

TMC

14.7%

BPSH

Changing patterns and differences of susceptibility between
both IMP resistant and sensitive isolates was observed. Most
of the isolates which showed resistance to imipenem and
either was MBL positive or negative were found highly resistant to other antibiotics compared to isolates which were
sensitive to imipenem. It was found that the resistance rate
among IMP resistant isolates toward ceftazidime was (69%),
piperacillin/tazobactam (66.6%), and cefepime (69%) respectively as illustrated in Table (4) compared to sensitive isolates.
Differences in resistance patterns between both hospitals are
shown in table 4.

Discussion
In the last few years MBLs have been identified from clinical
isolates worldwide with increasing frequency over the past
few years [7, 8, 17]. Strains producing these enzymes have
been responsible for prolonged nosocomial outbreaks that
were accompanied by serious infections [16]. The occurrence
of an MBL-positive isolate in a hospital setting possesses a
therapeutic problem, as well as a serious concern for infection
control management [17]. For that reason accurate identification, detection and reporting of MBL-producing P. aeruginosa will aid infection control practitioners in preventing the
spread of these multi-drug-resistant isolates [18].
The results of this study showed that P. aeruginosa isolates
were highly resistant to most usable anti pseudomonal antibiotics in both hospitals particularly for carbenicillin, cefotaxime,
piperacillin and ticarcillin. These findings are similar to other
studies reported worldwide [19]. Further higher resistance
rates were observed in BPSH for many anti pseudomonals
such as carbenicillin, cefepime, ceftazidime, gentamicin,
piperacillin, ticarcillin and tobramycin. The high incidence of
resistance at BPSH may be attributed to fact that many of anti
pseudomonal drugs were introduced long time ago in this
hospital than the TMC. Furthermore, there are an intensive
use of these antibiotics among patients attending this hospital and high prevalence of these bacteria in burn patients.
The results of this study is in consistence with other studies
reported the increasing incidence of P. aeruginosa resistance

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Table 3. (%) Resistant pattern of MBL positive and negative isolate strains from both TMCH and BPSH.
BPSH
N=23

TMCH
N=18

TMCH &BPSH
N=41

Hospital

N=2

P=21

N=5

P=13

N=7

p=34

Antibiotic

100%

100%

100%

100%

100%

100%

Imipenem (10)

100%

85.7%

100%

69%

100%

79.4%

Meropenem (10)

66.6%

66.6%

100%

61.5%

87.5%

64.7%

Ceftazidime (30)

66.6%

76%

60%

61.5%

62.5%

70%

Pip/tazobactam (110)

66.6%

80.9%

100%

53.8%

87.5%

70%

Cefepime (30)

66.6%

53.3%

40%

46.1%

50%

50%

Aztreonam (30)

100%

80.9%

100%

76.9%

100%

79%

Cefotaxime (30)

100%

90.4%

60%

76.9%

75%

85.2%

Ticarcillin (75)

66.6%

61.9%

60%

46.1%

62.5%

55.8%

Amikacin (30)

100%

85.7%

60%

46.1%

75%

70.5%

Pipercilline (100)

100%

61.6%

60%

46.1%

75%

55.8%

Tobramycin (30)

100%

85.7%

60%

46.1%

75%

58.8%

Ciprofloxacin (15)

66.6%

61.9%

60%

53.8%

62%

55.8%

Gentamicin (200)

0%

9.5%

20%

7.6%

12%

8.8%

Polymyxin B (300)

100%

95.2%

40%

76.9%

62.5%

88.2%

Carbenicillin (100)

Table 4. (%) Susceptibility pattern of IMP resistance and sensitive strains to anti-pseudomonas drugs.

4

BPSH

TMCH

TMCH&BPSH

n=142

n=170

n=312

Hospital

IMPS
118

IMPR
24

IMPS
152

IMPR
18

IMPS
271

IMPR
41

Antibiotics potency
(µg)

0%

100%

0%

100%

0

100%

Imipenem (10)

9.5%

62.5%

1%

77.7%

4.4%

69%

Meropenem (10)

32.2%

70.8%

14.4%

61.1%

22.2%

69%

Ceftazidime (30)

29%

75%

12.5%

61.1%

20%

66.6%

Pip/tazobactam(110

26.2%

75%

12.5%

61.1%

18.5

69%

Cefepime (30)

30.5%

58%

7.8%

44.4%

17.7%

61.9%

Aztreonam (30)

55.0%

66.6%

34.2%

83.3%

43.3%

73.8%

Cefotaxime (30)

69%

66.6%

34.2%

72.2%

49.6%

69%

Ticarcillin (75)

23.7%

62.5%

12.5%

50%

17.4%

57.1%

Amikacin(30)

44.0%

62.5%

18.5%

50%

29.6%

59.5%

Pipercilline (100)

35.5%

66.6%

14.4%

55%

23.7%

14.4%

Tobramycin (30)

30.5%

70.8%

13.1%

50%

20.7%

13.1%

Ciprofloxacin (15)

43.2%

45.11%

27.6%

55%

34.4%

50%

Gentamicin (200)

23.7%

8.2%

9.2%

22.2%

15.5%

14.4%

Polymyxin B (300)

53.3%

70.8%

35.5%

61.1%

43%

66.6%

Carbenicillin (100)

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in Burn patients to many antibiotics worldwide [20]. An early
study conducted by Ellabib [21] on infection in burn patients
showed that P. aeruginosa was the second leading pathogen
in the BPSH and was highly resistant to most early anti pseudomonal drugs such as cefotaxime, carbenicillin, piperacillin
and gentamicin. This study showed that all isolates were sensitive to ceftazidime, imipenem and aztreonam. These results
are also similar somewhat to the findings of Ellabib and colleges at TMC [22]. Our study clearly demonstrated that there
is an increase in the resistance pattern toward the new anti
pseudomonal antibiotics used in this investigation such as aztreonam, Cefepime, ceftazidime, ciprofloxacin and piperacillin/tazobactam. These findings are similar to the findings of
Shahid and co-authers who reported the incidence of AmpC
enzymes responsible for the resistance of Indian isolates of
P. aeruginosa [23]. Prevalence of MBL enzymes in our study
showed that the incidence over all was (10.8%), this finding
is in coherence with the findings of Pitout and co-authers [9].
Imipenem is recommended to test the occurrence of MBLs
[24]. The results of MBL detection using EDTA combined disc
were statistically significant for the three antibiotics used and
recommended for such study, and this finding were in agreement with the results reported by [25].
For most antibiotics, pattern of imipenem resistant isolates
positive to MBLs were higher in both hospitals compared
to MBL negative isolates for most antibiotics. Resistance to
ticarcillin, cefotaxime, cefepime, carbenicillin, ceftazidime and
piperacillin/tazobactam were found between (70 and 90%)
with some. These findings are due to the fact that resistant
isolates due to MBL production usually possess multiple resistant to all β-lactam antibiotics apart of the azetronam as a
result of the broad spectrum hydrolysis of these enzymes to
inactivate β-lactam antibiotics [1]. Furthermore, these resistant isolates may also harbor other mechanisms of resistance
to other classes of antibiotics such as; quinolones and aminoglycosides [1, 23]. Resistance to polymyxin B in this study was
the lowest (13.8%) and was similar to other studies [18]. This
is due to the mode of action of this antibiotic, which works
at the level of cell membrane affecting its integrity as well as
its limited external use [26].
Resistance of P. aeruginosa to the antibiotics (imipenem,
meropenem and ceftazidime) used in this study compared
with the sensitive isolates gives evidence to the occurrence
of MBLs in addition to the other test results that confirmed
such incidence. Our results showed that imipenem resistant
isolates were highly resistant to all anti-pseudomonal drugs
used in this study compared to sensitive ones. This is due to
the remarkable ability of P. aeruginosa to acquire resistance
mechanisms to many antimicrobial agents [28]. Such resistance mechanisms include chromosomal-mediated antibiotic

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Vol. 2 No. 1:3
doi: 10.3823/1014

resistance genes, also more importantly to plasmid-mediated
antibiotic resistance genes, class 1 integrons, transposons,
insertion sequences, Efflux pumps and outer membrane proteins that may play an important role in such resistance to
antibiotics [27, 28, 29]. Higher prevalence of such resistance
mechanism was observed in BPSH compared to that found in
TMC. It is not surprising that resistant isolates of P. aeruginosa
were found as nosocomial pathogens in the hospital setting
but the most interesting part of this study is the evidence
of occurrence of high percentage of MBLs in isolates from
patients admitted to intensive burn care unit as has been
reported by Haung and co-workers in 2006 [30].

Conclusion
The rapid dissemination of MBL producers is worrisome and
necessitates the implementation of surveillance and molecular genetic studies, and also to encourage the prudent use
of antibiotics, in particular carbapenems which are very effective drugs to treat life threaten pseudomonas infections.
The most active b-lactam antibiotic against MBL producing
P. aeruginosa isolates was polymyxin. The study showed that
MBL screening tests are simple and easy to perform as routine diagnostic tests for the detection of MBL production.
DDST is easier and less expensive than other MBL screening
tests such as E-test. The MBL-producing P. aeruginosa isolates
were more resistant to various antimicrobial agents and were
more prevalent in urine and wound cultures compared to
other samples.
We recommend that all imipenem and meropenem non
susceptible or -resistant P. aeruginosa isolates be routinely
screened for MBL production using the EDTA disk screen test
as described in this study. Finally routine detection of MBLs
will ensure optimal patient care and introduction of appropriate infection control procedures. Laboratories in Libya need
to improve qualit control policies, introduce recent standard
operating procedures (SOPs) and follow the international
guidelines for detection of antimicrobial resistance in bacteria.

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