Introduction:
P. aeruginosa and Acinetobacter species are organisms commonly causing nosocomial
infections and often associated with a resistant antibiotic profile. Carbapenems are the
the antibiotic of choice for treatment of infections caused by cephalosporin resistant
gram negative bacilli due to their broad spectrum of activity and stability to hydrolysis by
most beta-lactamases. This scenario is now changing with the emergence of metallobeta-lactamase (MBL) producing strains, especially among non fermenting Gramnegative bacilli (NFGNB) like P.aeruginosa and Acinetobacter species.[1,
2]
Resistance
to carbapenems is due to any one of the following mechanisms: decreased outer
membrane permeability, increased efflux systems, alteration of penicillin binding
proteins and production of carbapenem hydrolyzing enzymes (Carbapenemases).
According to Bush, Jacoby and Medeiros molecular classification carbapenem
hydrolyzing enzymes are classified into four groups A, B, C and D. MBLs belong to
group B and are enzymes requiring divalent cations as cofactors for their activity. MBLs
are inhibited by metal ion chelators such as CuCl3, FeCl3, EDTA, sodium
mercaptoacetic acid (SMA), mercaptopropionic acid (2MPA) and 2-mercaptoethanol
(2ME) but not by clavulanic acid, sulbactam or tazobactum. The MBLs efficiently
hydrolyze all β-lactams, except aztreonam in vitro. [3] Thus aztreonam is the treatment of
choice for infections caused by MBL producers. The IMP and VIM genes responsible for
MBL production are horizontally transferable via plasmids and can rapidly spread to
other bacteria. [4]
Although there are various methods recommended for screening MBL
production currently there are no guidelines recommended by the central laboratory
standards institute (CLSI). Commonly used for detection of MBL production are
Modified Hodge test (MHT)
(DDST)
[6],
[5],
imipenem/ceftazidime-EDTA double disc synergy Test
impregnated imipenem (IMP) discs
[7]
and minimum four-fold reduction in
MIC values with imipenem EDTA combination. [8]
It is important to use simple and reliable tests for detection MBL production
among clinical isolates of gram negative bacilli. This will help in appropriate treatment of
patients especially in intensive care settings and to control the spread of antibiotic
resistance. Therefore this study was undertaken to assess the local burden of MBL
production among NFGNB and to evaluate three phenotypic tests used for MBL
detection.
Materials and methods: This study was undertaken in the Department of Microbiology,
St.John’s Medical College, Bangalore from January to May 2009 during which a total of
8638 gram negative bacilli were isolated from various clinical samples such as wound
exudates, blood, CSF, urine, body fluids, respiratory secretions etc. 742 (8.58%) of
these were NFGNB. 116/742 (15.6%) of NFGNB were multi drug resistant. 100
consecutive non repetitive isolates of NFGNB resistant to imipenem (10µg) and
ceftazidime (30µg) isolated from clinical samples of patients admitted to this tertiary
care referral centre were included in the study. NFGNB were speciated using standard
phenotypic and biochemical tests.[9] Antibiotic susceptibility was tested by Kirby Bauer
disc diffusion method in accordance with CLSI guidelines, incorporating standard strain
of P. aeruginosa ATCC 27853 and E.coli ATCC 25922 for quality control.[10,11] The
antibiotics tested were piperacillin (100μg) gentamicin (10μg), amikacin (10μg),
netilmicin (30μg), ciprofloxacin (10μg), cefoperazone (75μg), ceftazidime (30μg),
carbapenems
[imipenem
(10μg),
meropenem
(10μg)],
polymyxin
B
(300U),
piperacillin/tazobactam (100/10μg) and aztreonam. MBL production was suspected
when the isolate was resistant to ceftazidime (CZD) and/ or imipenem (inhibition zone
diameter <18 mm for CZD and <16 mm for IMP). IMP resistance was confirmed by
determining the MIC by agar dilution according to CLSI guidelines.[11] (IMP
concentrations ranging from 0.062-128 μg/ml were tested). Isolates with imepenem MIC
of <4 μg/ml and >16 μg/ml are considered to be sensitive and resistant respectively.[11]
IMP resistant strains were screened for MBL production by MHT and
IMP-EDTA disc synergy test. DDST was done according to Lee et. al
IPM-EDTA disc method of Yong et al
[7]
[6]
modification of
by adding zinc sulphate to the medium to
increase the sensitivity of the test. Migliavacca et. al. [8] reported the use of microdilution
method for determining a fall in MIC values with IMP-EDTA combination, but we used
agar dilution method for the same.
Modified Hodge test (MHT): uses E.coli ATCC 25922 and a 10 μg IMP disc
instead of S.aureus ATCC 25923 and a 10 μg penicillin disc used in Hodge test.
[5,12]
A
lawn culture was made on Muller Hinton Agar (MHA) using overnight culture suspension
of E. coli ATCC 25922. (opacity of the tube adjusted by comparing with a 1:10 dilution of
0.5 McFarland opacity standard). After brief drying, a 10 μg IMP disc was placed at the
center of the plate and test strains were streaked from the edge of the disc to the
periphery of the plate in four different directions. Following overnight incubation, MBL
positive isolates showed a ‘clover leaf pattern’ in the zone of inhibition.
IMP-EDTA DDST
[6,7]:
Three modifications of this test were evaluated for
sensitivity in detection of MBL. An overnight broth culture of the test strain, (opacity
adjusted to 0.5 McFarland opacity standard) was lawn cultured on a MHA plate. In the
first modification a 10 μg IMP disc and a blank filter paper disc (6 mm diameter,
Whatmann filter paper no. 2) were placed 2.5 cms apart centre to centre. 10 μl of 0.5 M
EDTA (Sigma, USA) solution was added to the blank disc, (approximately 1.5 mg/disc).
In the second modification IMP and EDTA discs were placed touching each other edge
to edge. In the third modification two IMP discs 10 (µg) were placed on the surface of
an inoculated MHA agar plate and 10 μl of 0.5 M EDTA solution was added to one of
the discs. The zone of inhibition around both the IMP discs was recorded and compared
following 16-18 h incubation at 350C. An increase in zone size of at least 7 mm around
the IMP-EDTA disc is suggestive of MBL production.
0.5M EDTA solution was prepared by dissolving 186.1 g of disodium
EDTA.2H2O in 1000 ml of distilled water and adjusting it to pH 8.0 by using NaOH. The
mixture was sterilized by autoclaving. For long term usage EDTA impregnated IMP
discs were dried in an incubator and stored at 4oC and -20oC in airtight vials without
desiccant.
MIC of imipenem EDTA combination[8]: MIC of imipenem EDTA combination
was determined by agar dilution method. 1ml of EDTA solution was added to 1 ml of the
imipenem solution spanning similar concentrations as done for MIC to imipenem alone.
Each 2 ml of EDTA and imipenem in graded concentrations was added to 18 ml of
molten MHA and poured on plates that were allowed to set. A fixed inoculum of the test
strains was spot inoculated on the agar. The reading was taken after 18-24 h of
incubation. Highest dilution inhibiting the growth of organisms was recorded as the MIC.
Results:
100 consecutive NFGNB isolates resistant to IMP (10µg) and CAZ (30µg) by
disc diffusion method were obtained from clinical samples. (One isolate per patient) as
shown in Figure 1. 70% of the isolates were from critically ill patients admitted in
medical and surgical intensive care units. 66 of these were P.aeruginosa and 34 were
Acinetobacter species (Acinetobacter baumanii complex 30, Acinetobacter lwofii 4).
Antibiotic resistance in these isolates was as follows: 100% resistance to piperacillin,
gentamicin, CAZ, cefaperazone, imepenem and meropenem, amikacin (73%), netilmicin
(75%), ciprofloxacin (88%), cotrimoxazole (96%), piperacillin/tazobactam (48%). All the
isolates were sensitive to polymyxin B and aztreonam. Of the 100 NFGNB resistant to
IMP by disc diffusion, 93 had MIC values in the resistant range (8-128 µg/ml) and seven
showed intermediate resistance (MIC between 4-8 µg/ml). Results of MBL detection by
the three phenotypic methods is summarized in Table 1. 55 isolates were MBL
producers by MHT (Figure 2). In addition to the above 55 isolates IMP-EDTA DDST
detected MBL production in an additional eighteen isolates (Figure 3). The three
modifications of the DDST test detected MBL production in 65, 67 and 73 isolates
respectively.
8-128 fold reduction of MIC with imipenem EDTA combination was
observed in 74 isolates. P.aeruginosa ATCC 27853 and E.coli ATCC 25922 neither
exhibited a zone size enhancement with EDTA nor a fall in MIC. Potency of EDTA
impregnated IMP discs stored at 40C and -200C was checked every week. Discs stored
at -200C retained potency upto 16 weeks.
Discussion:
MBL was first reported as a zinc dependent enzyme in Bacillus cereus in
mid 1960s. [13] Few decades later, imipenem hydrolyzing metallo enzymes were found in
Aeromonas hydrophila[14] and Bacteroides fragilis.[15] All these enzymes were produced
by chromosomal genes and at first were recovered only from single clinical isolates. In
1991, Japan reported the first plasmid-mediated MBL in P. aeruginosa.[16] Apart from
P.aeruginosa, MBL production was also seen in other bacteria such as Serratia,
Klebsiella pneumoniae, Escherichia coli, Enterobacter aerogenes, Enterobacter
cloacae, Citrobacter freudii, Proteus vulgaris and Acinetobacter.[17]
In this study MHT detected MBL production in 55/100 imepenem resistant
NFGNB. EDTA impregnated IMP disc was able to identify MBL production in 73 isolates
(an additional 18 isolates). MIC to IMP-EDTA combination detected MBL production in
74 isolates. Studies across India report rates of MBL production ranging from 72%100% among carbapenem resistant NFGNB. The first report of MBL production in India
was in 2002 from urban hospital in Bangalore[18] which reported MBL production in all
the 6 (100%) carbapenem resistant P.aeruginosa. In 2005 Hemalatha V et al
[19]
reported MBL production among 87.5% of carbapenem resistant P.aeruginosa and all
the MBL producers were resistant to fluoroquinolones and aminoglycosides. From south
India Jesudasan et al[20] reported MBL production in 72% of imepenem resistant
NFGNB with concurrent resistance to aminoglycosides like Gentamicin, amikacin and
the quinolones which is similar to the findings in our study. Gupta V et al [21] reported
MBL production in 86.11% of imepenem resistant NFGNB using imepenem EDTADDST method. Varaiya et al
[22]
recorded MBL production in 83.3% carbapenem
resistant P.aeruginosa
isolated from ICU patients with 100% resistance to
aminoglycosides and cephalosporins. A study from rural India
[23]
reported imepenem
resistance of 8.62% among P.aeruginosa and 93.3% of these were MBL producers with
100% resistance to all the antibiotics tested. This high rate of MBL production among
P.aeruginosa in rural settings is alarming.
In our study the rate of MBL production among carbapenem resistant
NFGNB is 74%. The likely reasons for carbapenem resistance among non MBL
producers be varied such as, decreased cell membrane permeability or activity of efflux
pumps.[24] Predominant NFGNB showing imipenem resistance was P. aeruginosa, with
majority being isolated from respiratory samples like BAL, tracheal trap and sputum of
patients admitted in intensive care units. MBL producers in this study showed 100%
resistance to piperacillin, Gentamicin, ceftazidime, cefaperazone and 48% were
resistant to piperacillin- tazobactum which is lower than the findings of Navneeth et al [18]
and Chaudhray et.al
[25]
who have reported 100 per cent resistance to piperacillin-
tazobactum. However in this study all the MBL producers were sensitive to polymyxin B
(300U) and aztreonam. Recently studies have shown that polymyxins are not as toxic
as previously thought
26]
and thus there is a renewed interest in the use of non-
traditional agents including polymyxin B and E (colistin) for the treatment of serious
infections caused by MDR P. aeruginosa. However monotherapy with these agents
should be avoided and combination therapy with a sensitive aminoglycoside or
fluroquinolone is preferred. In the absence of a novel antibiotic agent in the near future
polymyxin B should be used judiciously. As CLSI does not provide guidance for
susceptibility testing of polymyxins, serum bactericidal assays become prudent to
regulate the dosage when these agents are used. There are reports on MBL production
in P.aeruginosa from countries across the globe such as Brazil [27], Korea [28], Singapore
[29]
and France [30]. There are reports of MBL production in P. aeruginosa from the Asian
and the Pacific countries, namely Hong Kong, Taiwan and Japan
[31] as
well.
Although there are several screening methods used and recommended for the
detection of MBL production, currently there are no CLSI guidelines for the detection of
these enzymes. MHT is a simple test for screening for MBL production but occasional
false negative results have been reported.[6] This could be avoided by incorporating zinc
sulphate in a final concentration of 70μg/ml into the MHA.[6] In this study EDTA
impregnated IMP disc showed a better sensitivity in the detection of MBL which is
similar to the observations of other workers. [6, 20] 8-128 fold decrease in MIC values with
imipenem EDTA combination was observed in this study as compared to a 4-512 fold
reduction as reported by Migliavacca et al.[8] Other methods such as PCR
[33]
[32]
and E test
have been used to identify MBL producers. However, these tests may not be cost-
effective for routine testing in clinical laboratories. PCR has become more difficult with
the increasing number of types of MBLs.[7]
Conclusion: For infections caused by MBL producing NFGNB therapeutic options are
severely limited. Therefore identification of MBL producers is of great importance for the
appropriate treatment of these infections and also to control the spread of resistance.
Thus a simple screening test is essential to monitor resistance. The two screening tests
for MBL detection namely, IMP-EDTA DDST and MIC to IMP-EDTA combination are
equally effective. Based on the findings of this study we conclude that for MBL detection
EDTA disk synergy test is more sensitive than MHT and much simpler than the MIC
detection for imepenem EDTA combination. With the stored EDTA discs having a shelf
life of upto 16 weeks this is a convenient method in a regular bacteriology laboratory to
monitor the production of MBLs and for antibiotic resistance surveillance. However we
could not include a standard ATCC carbapenem resistant positive control in the study
since none is available for Nonfermenting gram negative bacilli and there is no
standardized methodology recommended by CLSI.
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Figure 1: Shows the clinical samples from which NFGNB included in the study
were isolated.
DISTRIBUTION OF ISOLATES FROM CLINICAL SAMPLES
CLT
1
1
CLINICAL SAMPLES
DRAIN/ASCITIC FLUID
3
1
URINE
3
BLOOD
4
1
WOUND SWAB
7
13
2
PUS
9
1
ET TIP
4
2
SPUTUM
6
1
9
TREACHEAL TRAP
0
P.aeruginosa
A.baumanii
15
2
A.lwoffii
4
6
8
10
NO.OF SAMPLES
17
12
14
16
18
Figure 2: MBL production by modified Hodge test: Isolates A and B are MBL
producers showing a clover leaf pattern. Isolates C and D do not produce MBL. Isolate
D is ATCC P.aeruginosa used for quality control.
Figure 3: MBL production using IMP-EDTA Double disk synergy test
Table1: Results of the three phenotypic tests for MBL detection
Test
No. of isolates
positive for MBL
Sensitivity Specificity PPV†
NPV‡
P.aeru Acineto
ginosa -bacter
spps.
MIC with
IMP +EDTA
49
25
100%
100%
100%
100%
DDST
48
25
98.6%
100%
100%
96.42%
MHT
36
19
74.32%
97.1%
98.21%
60%
(Sensitivity and specificity in comparison to MIC)
†PPV- positive predictive value, ‡ NPV- negative predictive value