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Nosocomial outbreak of carbapenem-resistant
Pseudomonas aeruginosa producing metallo
beta lactamase in Al-Hilla teaching hospital,
Babylon
Eman M. Jarallah Mustafa J. Alammary
Abstract
Metallo-beta-lactamase MBL producing Pseudomonas aeruginosa have been reported to show
resistance to carbepenem drugs. Detection of metallo-beta-lactamases producing P. aeruginosa is now
important to prevent their spread as dissemination of these bacteria could be fatal to the patients.
The present study was undertaken over period of six months from December-2011 to May-2012 in Alhilla
teaching hospital, to study the incidence of outbreak of MBL producing P.aeruginosa from
various clinical specimens. Out of 247 clinical specimens 81 isolates were obtained . These isolates
were subjected to susceptibility testing to antipseudomonal drugs as per CLSI guidelines, 32(40%) of
isolates were carbapenem resistant P. aeruginosa (CRPA). They were further screened for production
of MBL: by EDTA-imipenem synergy method and Carbapenemase production: by modified Hodge test
and using E. coli ATCC 25922 for control.
All (CRPA) isolates were MBL producing isolates and carbapenemase producing isolates.
1. Introdution:
Pseudomonas aeruginosa is a frequent nosocomial pathogen that causes a wide
range of opportunistic infections and nosocomial outbreaks [Hammami et al.,2011].
Infections by P. aeruginosa are a serious clinical problem, particularly in immune
compromised hosts in hospital settings . Moreover, the treatment of these infections is
often difficult because of the limited number of effective antimicrobial agents, due to
the intrinsic resistance of P. aeruginosa strains and their different modes of growth
[Martinez et al.,2011]. Carbapenem ,such as imipenem, meropenem remain one of the
best drugs to treat infections caused by p. aeruginosa , but increasing usage of these
drugs and other expanded-spectrum antibiotics has resulted in the development of
carbapenem –resistant p. aeruginosa. [Vitkauskiene et al.,2011;Sacha et
al.,2007;Pournaras et al.,2007;Yousefi et al.,2010;Martinez et al.,2011]. The common
form of resistance is mediated by lack of drug penetration(i.e.,porin mutations and
efflux pumps) and /or carbapenem hyrolysing beta lactamase enzymes including the
metallo beta lactamases (MBL).[ Shanthi and Sekar.,2009;Ishii et al.,2010]. P.
aeruginosa is able to acquire (MBL) by horizontal transfer of mobile genetic elements
coding for class B carbapenemases (also called metallo-?-lactamases [MBLs]), which
hydrolyze all ?-lactams except aztreonam [Viedma et al.,2012; Gutierrez et
al.,2007;Horii et at.,2007]. The IMP and VIM genes responsible for MBL production
are horizontally transferable via plasmids and can rapidly spread to other bacteria , the
dissemination of which contributes to the global spread of this resistance mechanism
[Zubair et al.,2011; Viedma et al.,2012; Varaiya et al.,2008]. Carbapenemases can be
classified into two main molecular families: those with serine at their active site,
known as serine carbapenemases, and those with at least one zinc atom at their active
site, known as metallo-carbapenemases which are a subgroup of metallo beta
lactamases (MBLs)[Bahar et al.,2010]. So, (MBLs) require divalent cations of zinc as
co-factor for enzymatic activity and are universally inhibited by ethylenediamine tetra
acetic acid (EDTA), as well as other chelating agents of divalent cation[Kumar et
al.,2012;Bratu S. and Quale J., 2006]. It found that the tests of modified hodge and
EDTA-imipenem Doudle disk synergy were simple to use the screen metallo-Blactamase
producina strains of aeruginosa[Lee et al., 2003] . Detection of (MBL)
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producing P. aeruginosa is crucial for optimal treatment of patients particularly
critically ill and hospitalized patients and to control the spread of resistance[Bashir et
al.,2011] .The aim of this study was to determine the occurrence of MBLs among
carbapenem-resistant P.aeruginosa clinical isolates in Al-Hilla teaching hospital.
2. Materials and methods:
2.1. Patients and bacterial strains:
This study included clinical isolates of carbapenem resistant P.aeruginosa
(CRPA) from patients of Al-Hilla teaching hospital in Babylon city , Iraq , During the
period from December 2011 and May 2012. A total of 247 sample included burn
swab, ear swab, wound swab, urine & blood culture and bed-patient room, were
cultured on blood agar and MacConkey agar plates and incubated at a temperature of
37 °C for 24 h on Mueller Hinton agar plates in order to to assess pigment
production. The culture plates were processed using standard microbiological
procedures [Olayinka et al.,2009].
Characterization and identification of P. aeruginosa was carried out using a
combination of colonial morphology, Gram stain characteristics, motility tests,
pigmentation, oxidation-fermentation tests, catalase and oxidase activity tests and
pyocyanin production [Paranjothi S and Dheepa R., 2010].
2.2. VITEK 2 system:
The newly launched VITEK 2 Compact, a system for identification and
antibiotic susceptibility testing (AST) [Fig.1]. VITEK2 Compact is designed for
medium to small-sized laboratories which require an automated system that will cover
the majority of their routine tests [BioMérieux ] .This system that produced by
BioMérieux company used in this study to identification of bacterial isolates with
probability 98% and to determine the MIC of antibiotic to bacterial isolates.
2.3. Antimicrobial susceptibility test :
Antimicrobial susceptibility was tested using the microdilution method, with
Mueller-Hinton agar (HIMEDIA,India). The following antimicrobials were tested:
ampicillin piperacillin, gentamicin, tobramicin, amikacin, ciprofloxacin, ceftazidime,
cefepime, imipenem, meropenem, polymixinB [Martinez et al.,2011]. The antibiotics
were purchased from (HIMEDIA Co.,India). All P. aeruginosa strains were screened
for resistance to carbapenems(IMP and MEM) by the routine disk diffusion test with
13-15mm inhibition zone according to the guidelines of the Clinical and Laboratory
Standards Institute (CLSI). E.coli ATCC 25922 was used as control strain.
2.4. Phenotypic detection of metallo-b-lactamase[MBLs] production:
In this study, IMP-EDTA-disk synergy test developed by [Yang et al.,2002]
was used for detection of [MBLs] P.aeruginosa isolates . we prepare 0.5M EDTA
solution and sterilized by autoclave. EDTA-IMP-synergy disk prepare by adding
EDTA solution to IMP 10 disk and put the disk on Mueller Hinton agar , in the same
disk we put only IMP disk as control. After overnight incubation, the established
zone diameter difference of less than 7mm between EDTA-IMP-synergy disk and
IMP only disk was interpreted as positive result of [MBLs] production[Irfan et
al.,2008] [Fig. 2].
2.5. The Modified Hoge test [MHT]:
Carbapenemase production was detected by the [MHT] when the test isolate
produces the enzyme(carbapenemase) and allows growth of a carbapenem susceptible
strain (E.coli ATCC 25922) towards a carbapenem disk. An overnight culture of
indicator organism E. coli ATCC 25922 was adjusted to a turbidity of McFarland No.
0.5 and No. 0.05 and these were used to swab inoculate the surface of the agar plates.
After drying the surface, test organisms were heavily streaked from the center to the
Journal of Babylon University/Pure and Applied Sciences/ No.(1)/ Vol.(22): 2012
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periphery of the plate using an inoculating loop and a 10-?g imipenem or meropenem
disk (HIMEDIA) was placed at the center, and incubated overnight. The Hodge test is
interpreted as positive by the presence of distortion of the inhibition zone The result is
a characteristic clover leaf-like indentation [Dugal S. and Fernandes A., 2011; Lee et
al.,2010] [Fig.3].
3. Result and Discussion:
3.1. Bacterial strains isolation ; A total of 247 clinical samples collected from
different sources of patients admitted in Al-Hilla teching hospital, 81(33%) of
samples was P.aeruginosa isolates. Most isolates was identified by morphological
shape of colonies that cultured on blood agar and MacConky agar, in addition to
pyocyanin pigments producing on Mueller Hinton agar. Biochemical tests such as
oxidase & catalase production, IMVIC tests ,urase test and TSI fermentation test.
VIEK 2 system used for more accuracy identification and for MIC test for
antibacterial agents.
3.2. Antimicrobial susceptibility test : out of 81 P.aeruginosa isolate, 32(40%) of
isolates was resistant to carbapenem [imipenem 13 (16%), meropenem 19 (23%)]
antipseudomonas agent with inhibition zone less than 16mm by using disk diffusion
method. Isolates resistant to carbapenem were also resistant to other antibiotics:
52(64%) amikacin, 69(85%)gentamicin, 44(54%)pipracillin, 73(90%) ampicillin,
39(48%) ciprofloxacin, 49(60%) cefepime, 67(83%)ceftazidime, 62(77%)tobramicin,
75(92%) polymixin B [Table.1].
3.3. VITEK 2 system: three isolates tested by VITEK 2 system[BioMérieux ] because
of cost of cards. ( 98% ) probability of P. aeruginosa identification after 5 hours
analysis. . All isolates was resistant to 13 antimicrobial agent applied in this system.
Fig. 1. VITEK 2 Compact system [BioMérieux ] for identification and antibiotic
susceptibility testing (AST)
3.4. metallo beta-lactamase production: EDTA-IMP- synergy disk used for detection
of metallo-beta lactamase production isolates. Inoculums prepared equal to 0.5
McFarland(equivalent to 1.5 x 108 cfu/ml) and cultured on Mueller Hinton agar, put
EDTA-IMP disk and IMP disk alone and incubated overnight. all carbapenem
resistant isolates 32 (100%) showed metallo beta lactamase activity with an average
differences in inhibition zone 17mm between EDTA-IMP disk and IMP disk alone.
Carbapenem sensitive isolates did not show any metallo beta lactamase activity
[Fig.2].
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Fig. 2. EDTA-IMP-synergy disk and IMP only disk was interpreted as positive result
of [MBLs] production.
3.5. modified Hoge test: carbapenemase production isolates detected by this test.
E.coli ATCC 25922 used as control to differentiation between carbapenem sensitive
strains[E.coli ATCC 25922] and carbapenem resistant strains
[P.aeruginosa isolates]. All of carbapenem resistant isolates reach to IMP or MEM
disk in center while E.coli ATCC 25922 show inhibition zone to forming clover leaflike
shape [Fig.3].
Fig. 3. Modified Hoge test for carbapenemase producing P.aeruginosa isolates
detection.
3.6. Epidemiological data: All of 247 sample had been collected and directly cultured
from December 2011 to May 2012. Different sources of study samples included out of
247 sample 107(43%) burn swabs, 34(14%) wound swabs, 33(13%) ear swabs,
31(13%) urine samples, 15(6%) blood samples, 27(11%) bed and patient rooms. Most
of P.aeruginosa isolates was burn samples[Table. 2] . Because of the P.aeruginosa
was nosocomial infection, all samples collected from at least after one week from
patients admission.
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Table 1. susceptibility of P.aeruginosa isolates to antimicrobial agents according to
CLSI*:
Diameter of inhibition zone in mm with percentage of
isolates
Antimicrobial agents
Sensitive No. (%) Resistant No. (%)
Ampicillin 17 8(10%) 13 73(90%)
Amikacin 17 29(36%) 14 52(64%)
Gentamicin 15 12(15%) 12 69(85%)
Pipracillin 18 37(46%) 17 44(54%)
Ciprofloxacin 21 42(52%) 15 39(48%)
Cefepime 18 32(40%) 14 49(60%)
Ceftazidime 18 14(17%) 14 67(83%)
Tobramicin 15 19(23%) 12 62(77%)
Polymixin B 12 6(8%) 11 75(92%)
Imipenem 16 68(84%) 13 13(16%)
Meropenem 16 62(77%) 13 19(23%)
* Clinical and Laboratory Standards Institute (CLSI) 201
Table 2. Distribution of P.aeruginosa isolates among clinical specimens:
Source of samples Samples No.(%) P.aeruginosa
isolates No.(%)
Carbapenem resistant
isolates No.(%)
Burn swabs 107(43%) 54(67%) 29(91%)
Wound swabs 34(14%) 5(6%) 1(3%)
Ear swabs 33(13%) 3(4%) 0
Urine samples 31(13%) 7(9%) 0
Blood samples 15(6%) 3(4%) 1(3%)
Ded & patient room
swabs
27(11%) 9(11%) 0
Total 247(100%) 81(100%) 32(100%)
3.7. Discussion: Carbapenems are potent agents against multiresistant Gram negative
bacilli, including P. aeruginosa, but their efficacy is increasingly compromised by the
emergence and the worldwide dissemination of carbapenem resistance strains, which
are implicated in large outbreaks as described in many countries [Hammami et
al.,2011]. P.aeruginosa producing MBLs was first reported from Japan
in1991[Zubair et al.,2011]. The clinically important MBL families are located in
horizontally transferrable gene cassettes and can be spread among Gram-negative
bacteria [Bahar et al.,2010]. MBL producing isolates are associated with a higher
morbidity and mortality. Moreover given the fact that MBLs will hydrolyze all classes
of ?-lactams [such as carbapenems] and that we are several years away from the
development of a safe therapeutic antibiotic; their continued spread would be a
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clinical disaster. The occurrence of an MBL positive isolate poses not only a
therapeutic problem but is also a serious concern for infection control management
[Bashir et al.,2011].
In this study we were collected the sample after one week to isolate nosocomial
acquired P.aeruginosa so, all or most of 81 isolates was 54 burn patients isolates, and
susceptibility test show 29 CRPA isolates was burn swab samples, 1 wound swab and
1 blood culture sample. This result lead to that all carbapenem resistant isolates was
from burn patients admitted at Al-Hilla teaching hospital, this results were
Compatible to another study at Shahid Motahari level I burn hospital in Tehran [Bahar
et al.,2010], So we were interpreted the outbreak of MBLs producing bacteria in Iraq s
Hospitals causing by travelling of patient between Iran-Iraq hospital because of
horizontally transferrable gene cassettes [IMP & VIM] responsible on MBLs feature.
The spread of MBLs producing P.aeruginosa isolates should be considered an
emerging pandemic specially in Asia [Viedma et al., 2012].
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