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Detection of
metallo-
-lactamase in
Pseudomonas
aeruginosa
from Tripoli, Libya
3Department 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 Conventuals 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.
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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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--lactamase mediated resistance in P. aeruginosa have escalated since 2000
and severely affected treatment options in Asia, Europe and
Latin America to non--lactam antibiotics [8]. Clinical isolates
harboring metallo--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 -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).
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Results
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
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.
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
(g)
0%
100%
0%
100%
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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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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