‫‪1‬‬
‫‪Comparison of Two Different Methods for Detection‬‬
‫‪of Extended-Spectrum Β-Lactamase Producing‬‬
‫‪Klebseilla pneumoniae.‬‬
‫٭‪Jafaar K. AL- Mouswi PhD.‬‬
‫٭)‪Zina M. Al-Shami(Msc‬‬
‫٭٭‪Israa K. AL-yasiri Msc.‬‬
‫‪ Department of Microbiology, college of medicine, kufa university .‬٭‬
‫‪ Department of Microbiology, college of Dentistry, kufa university.‬٭٭‬
‫الخالصة‪:‬‬
‫ِعدّة اختبارات سريريه ابتكررت الكتشرا‬
‫البكتيريرا المنتجر نزرمي ألبيترا‪-‬الكتراييم ت َتضر ّمُ ذر‬
‫االختبرارات التركيرم المبرب اندزر ‪ ،‬اختبرار ازتشرار الصرر‬
‫‪ ،‬طريصر ‪، Iodometric‬و طريصر‬
‫يُ للكش ر ِ عررُ ‪K.‬‬
‫‪ ،Acidometric‬الدراس ر الحالي ر تحرراو المصَ َ‬
‫ارزَ ر برريُ طررريصتيُ يختلفت ر ِ‬
‫‪ pneumoniae‬المنتج نزمي ألبيتا‪-‬الكتا ييم الواسع الطي‬
‫وطريصرر‬
‫‪ :‬اختبار الصر‬
‫البنائي(‪)DDST‬‬
‫‪ Iodometric‬باإلضررا ىلرر ىيجرراد المسررا ِ المناسررب أكبررر لوضررع أنقرررا ِ رري‬
‫طريصر ِ (‪ .)DDST‬تر زصررد يحررددات المصاوير ِ ‪ِ ،‬يررُ ‪ - 6‬عترررات‬
‫‪ K. pneumoniae‬المنتجر‬
‫نزمي ألبيتا‪-‬الكتاييم ىل عمل قياسي ( ‪ )E.coli M 40203/1‬حساس لكد المضادات الحيوي‬
‫ث تل ذلك ىجراء حص قابلي ذ‬
‫البكتريا المصترز عل ىزتاج ىزرمي البيترا الكتراييم باسرتخدام‬
‫طريصر (‪ )DDST‬وطريصر ‪ . Iodometric‬أظهرررت النتررائد درجر عالير يررُ الحساسرري سررجل‬
‫بواسررط اسررتخدام طريص ر (‪) 100% ()DDST‬بالمصارز ر يررع اسررتخدام طريص ر ‪Iodometric‬‬
‫(‪.)66.67%‬وو صا لنتائد ذ االختبارات يبدو ىن طريصر (‪)DDST‬ذري الطريصر انكبرر عملير‬
‫وقبرروال لتحديررد البكتريررا المنتجرر لإلزررمي البيتررا الكترراييم الواسررع الطيرر‬
‫بالمصارزرر يررع طريصرر‬
‫‪ Iodometric‬والتي ظهر يُ خال النتائد أزها طريص اقد حساسي وذك ا إزها ليس عملي جدا‬
‫لالستعما‬
‫ي المختبرات ‪.‬‬
2
Summary:
Several clinical tests have been devised to detect ß-lactamases
producing
bacteria.
These
tests
include
Minimum
Inhibitory
Concentration, Disk Diffusion Test, Iodometric Method, Acidometric
Method, and Chromogenic Substrates. The present study try to compare
between two different methods for detection of extended-spectrum βlactamase (ESBLs) producing K. pneumoniae: Double-Disk Synergy disk
Test (DDST) and Iodometric method as well as to detect the most suitable
distance for the disks in DDST method. Resistance characterization was
done by transferring the resistance determinants from 6- ESBLs
producing K.pneumoniae
into a standard strain (E.coli M 40203/1)
resistant to Rifampicin followed by testing these strains {ESBLs
producing K. pneumoniae, E.coli M 40203/1 and the Transconjugant
strains} for the production of β-lactamase using both DDST and
Iodometric method. The results showed a high degree of sensitivity
recorded by DDST (100%) in comparison to the sensitivity of Iodometric
method (50%). According to the results of this investigation the DDST
seems to be a practical and acceptable method for detection of extendedspectrum β-lactamase (ESBLs) producing K. pneumoniae in comparison
to the Iodometric Method who showed to be a less sensitive method and
thus not very practical for use in routine work and clinical laboratories.
Key words: DDST, Iodometric method and ESBLs.
3
Introduction :
Multiple antimicrobial resistant pathogens are one of the major
causes of infectious diseases and hospital acquired infection world wide.
The importance of these bacteria has further increased by appearance of
extended-spectrum ß-lactamase (ESBLs) enzymes (Mugnaioli, et al.,
2006). The majority of ESBLs classified in Bush-Jacoby-Medeiros
group2 belong to the TEM and SHV families of enzymes (Bush, et al.,
1995). These enzymes hydrolyze the amide bond of the ß-lactam ring of
antibiotics including 3rd generations Cephalosporins and Aztreoname the
genes responsible for there production are plasmid mediated so they can
be transferred to other species of bacteria through conjugation and
transformation
of
plasmids
(Bradford,
2001).
Although
other
Enterobacteriaceae and Pseudomonads have been found to produce
ESBLs enzymes the organisms most likely to produce ESBLs are
Klebsiella pneumoniae (K. pneumoniae) (Xiong, et al., 2002).
Due to their global presence, plasmid-mediated ESBLs producing
K. pneumoniae became a matter of great concern in modern laboratory
researches. Right bacteriological diagnosis that helps in a fast detection of
ESBLs producing bacteria consider a good step in decreasing morbidity
and mortality rate caused by those pathogens (Xiong, et al., 2002). So the
present study was conducted to compare between 2-different diagnostic
methods (Double-Disk Synergy disk Test (DDST) and Iodometric
method) for detection of ESBLs producing K. pneumoniae and to
evaluate their significance in order to be recommended in routine
laboratory work.
4
Materials and Methods
Bacteria
The test organisms were 6 clinical strains of Klebseilla
pneumoniae. these strains were obtained from Al-Hakeem Teaching
Hospital during the period from May-2005 to March-2006 from various
clinical specimens. Isolation of pathogenic bacteria from clinical
specimens and identification to the species level was performed by
standard methods (McFadden 2000) and using of API-20 system. The
strains showed antibacterial resistance for many antibiotics specially 3 rd
generation Cephalosporins (Ceftazidime and Cefotaxime) so they were
screened for ESBLs production with screening and phenotypic
confirmatory tests recommended by the National Committee for Clinical
Laboratory Standards (NCCLS 2000). The strains who showed positive
results (6-strains of K. pneumoniae) were treated with the following tests
for confirmation their ESBLs production:
1-Transfer of resistance characters:
K. pneumoniae strains were investigated for the transfer-ability of
their resistance determinants in order to ensure their plasmid mediated
resistance. Conjugation experiments were set up employing a standard
strain (E.coli M 40203/1) resistant to Rifampicin.
Logarithmic phase Mueller-Hinton broth (BBL) cultures of the
donor (all the 6-tested strains of K. pneumoniae) and recipient strains
(E.coli M 40203/1) were mixed at a ratio of 1:2 of the donor and recipient
strains and incubated without shacking at 37˚C for 18h. Transconjugant
colonies were selected on MacConekey agar containing Ceftazidime
(2mg/L) and Cefotaxime (2mg/L). Frequency of transconjugation was
calculated relative to the number of cells appeared on MacConekey agar
containing Ceftazidime (2mg/L) and Cefotaxime (2mg/L) to investigate
the frequency of transformation.
5
2- Extended-Spectrum ß-Lactamase Production Tests:
A- Double-Disk Synergy Test (DDST):
The 6-strains of K.pneumoniae (S1, S2, S3, S4, S5 and S6)were cultured
in a Mueller-Hinton broth and standardized to an optical density equal to 0.5
McFarland turbidity and cultured on Mueller-Hinton agar plates. Oxoid
antibiotic disks including Ceftazidime (30µg), Ceftriaxone (30µg),
Aztreoname (30µg), and Cefotaxime (30µg) were placed 2.5cm and 3cm
apart from the central Amoxycillin-clavulanate (30-15µg) disk. A clear-cut
extension of the inhibition zone around the ß-lactam antibiotic disk towards
the Amoxycillin-clavulanate (30-15µg) containing disk was interpreted as a
positive double-disk synergy test for that particular ß-lactam. Any strain
shows obvious resistance to Cefotaxime in the presence of Amoxycillinclavulanate (30-15µg) was considered as Cefotaximeases producing (CTXM β-lactamases) strain.
B- Iodometric Method:
A loop full of over night grown culture (of the tested strains) was
transferred into small tube containing 1ml of penicillin G solution and
incubated at 37˚C for 30 min, 0.5ml of Starch Solution and 0.2ml of
Iodine Solution was added and mixed for 2-3min. the change of black
color to colorless, indicates a positive test (Bush et al.,1995).
Preparation of Iodometric method solutions:
1. Penicillin G Solution:
Prepared by dissolving 0.6g of penicillin G in 60ml of phosphate
buffer , the volume was made up to 100ml with Distilled Water (D.W.),
and sterilized by Millipore filters (0.22μm). (Bush et al.,1995).
6
2.Starch Solution
Prepared by dissolving 1g of starch in 100ml of D.W. using water bath at
100˚C for 10 min. (Bush et al.,1995).
3.Iodine Solution
Prepared by dissolving 2g KI in 25 ml of D.W. and 1g of iodine was
added and stirred vigorously for 15min and made up to 100ml with
D.W..(Collee et al., 1996).
Results And Discution :
ß-lactam antibiotics are among the safest and most frequently
prescribed antimicrobial agents worldwide. The emergency of resistance
to these agents appeared in the past 2-decades and resulted in a major
clinical crisis (Randegger and Hàchler, 2001). According to a survey by
the National Committee for Clinical Laboratory Standards the prevalence
of ESBLs producing Enterobacteriaceae range from an average of 3% in
United Stats to a much higher average in Europe in which 11.4% of
K.pneumoniae were found to be ESBLs producer (NCCLs, 2000).
To avoid the increasing threatening of ESBLs producing strains rapid
ß-lactamase tests can yield clinically relevant information for the most
suitable antibiotic therapy and can help in preventing further resistance
mutations .
ESBLs are plasmid-mediated ß-lactamases and can be transferred to
a non ESBLs producing bacteria depending on this theory the
transconjugation method was performed using a standard strain (E.coli M
40203/1) as an receipeint cells and the tested K.pneumoniae strains were
the donor cells this experiment was performed to detect genetically the
production of ESBLs enzyme (Table 1). The results showed that five of
the six strains were able to grow on MacConekey agar containing
7
Ceftazidime (2mg/L) and Cefotaxime (2mg/L) in different frequencies.
High frequency was recorded by strain S5 (32×10-9) in comparison to a
lower frequency recorded by strain S2 (4×10-9), while strain S3 failed to
grow.
Table 1: Frequency of Transconjugant Growth on MaCconekey
Agar Containing Ceftazidime and Cefotaxime .
Strain
S1+ E.coli M
40203/1
S2+ E.coli M
40203/1
S3+ E.coli M
40203/1
S4+ E.coli M
40203/1
S5+ E.coli M
40203/1
S6+ E.coli M
40203/1
Total
Growth on
MacConekey
agar
Frequency
+ve
23 ×10-9
+ve
4×10-9
-ve
0
+ve
27×10-9
+ve
32×10-9
+ve
11×10-9
5/6 (83.33%)
In order to compare between the viability of DDST and Iodometric
method the two methods were performed on the same strains (Table 2)
the results showed that DDST results were similar to transconjugation
results which showed that (83.33%) of the tested strains were ESBLs.
Transferring of resistance determinants (plasmids) from the tested
strains to (E.coli M 40203/1) strain who occurs in 83.33% of strains
confirms the sensitivity of DDST in detecting ESBLs production in
comparison to Iodometric method which recorded a lower frequency
50%.
8
Table (2): Comparison between DDST and Iodometric Method
in Detection of ESBLs Producing K.pneumoniae .
K.pneumoniae
Strains
ESBLs Production Tests
Results
DDST Results
Iodometric
Test
CTX-M βESBLs
lactamases Results
1
+ve
-ve
+ve
2
+ve
+ve
+ve
3
-ve
-ve
-ve
4
+ve
-ve
-ve
5
+ve
-ve
+ve
6
+ve
+ve
-ve
2/6
(33.33%)
3/6
(50%)
Total of +ve
5/6
results(Percentage) (83.33%)
DDST method depends on measuring of inhibition zone around the
disks and comparing it with those reported by the NCCLs, while the
reading of Iodometric method results depends on the visual way of
reading almost on color change that may cause error in reading of the test
results or doubtful results.
Similar results were recorded by A total of 20.8% isolates were
producers of b-lactamases as found by iodometric test method. On the
other hand, screening for the production of ESBL with ceftazidime disc
showed that 38.5% of the isolates were the producers of ESBLs. The
figure shows comparison of constitutive and ESBL production.
In concerning of DDST performing, it is important to put all four
disks (Ceftazidime , Cefotaxime, Ceftriaxone and Aztreonam) on the
plate and this is could be attributed to different causes first, Ceftazidime
9
and Aztreoname is more sensitive indicators for SHV-5 ß-lactamase
producers while Cefotaxime and Ceftriaxone are more reliable in
detecting SHV-2 ß-lactamase producers. Second, for the detection of
Cefotaxime (CTX)-resistant and/or Ceftazidime (CAZ)-resistant isolates
(Moubareck, et al., 2005).
During the study, tow strains (S6 and S2) were resistant to the effect
of Cefotaxime disk even in the presence of Amoxycillin-clavulanate (3015µg) disk. Depending on these results we can say that the two strains
(S6 and S2) could be from the Cefotaximeases (CTX-M β-lactamases)
group, a new family of plasmid-mediated ESBLs, these enzymes
preferentially hydrolyzes Cefotaxime and are more active against
Cefotaxime than Ceftazidime (Kariuki, et al., 2001). These enzymes have
been observed in members of the family Enterobacteriaceae since 1990
they are plasmids mediated enzymes and may be co-transferred with
ESBL genes to transconjugants through conjugation experiments to the
recipient strain, this indicates the possibility of horizontal transfer of
resistance gene (Mugnaioli, et al., 2006).
Detection of CTX – M strain that could be added to the DDST features
in which the ability of this test to further detection of CTX-M strains
make this test more specific. Similar results were recorded by Quinteros,
et al., 2003 who recorded that the DDST detected all SHV-5 SHV-12 βlactamase producers and 95.2% of SHV-2 so it was highly specific in
comparison to other tests used in their study.
Accurate distances between disks are important to give correct
results therefore, two distances were examined in DDST in order to find
out the most suitable distance for the 4-antibiotic disks from the central
disk to avoid false results resulted from distance variation. The four
10
antibiotic disks {Ceftazidime(30µg), Ceftriaxone(30µg), Aztreoname
(30µg) and Cefotaxime(30µg)} were placed in distances of 2.5cm and
3cm from the central Amoxycillin-clavulanate (30-15µg) disk. As shown
in (Table 3) there was a slight difference between the two distances.
Similar results were recorded by Quinteros, et al., 2003.
Table 3: The Sensitivity of DDST in 2.5cm and 3cm Distance
from the Central Disk.
Bacterial strains
2.5cm
Distance
3 cm Distance
No.
%
No.
%
K. pneumoniae
5/6
83.33
2/6
33.33
Transconjugant
strains
5/5
100
2/5
40
ESBL producing isolates are in increasing and CTX-M type ESBLs
producing strains begin to appear in Al-najaf Governorate, which might
be attributed to the wide use of third generation Cephalosporins,
especially Cefotaxime. So it is important to monitor and detect such
strains closely, and necessary measures must be taken to prevent their
spread. So it is recommended to use a fast and reliable method for
detection of ESBLs,
Cefotaxime (CTX)-resistant and Ceftazidime
(CAZ)-resistant isolates. in conclusion , and according to the results of
this study we can say that the DDST method (in comparison to the
Iodometric method) is a specific, reliable and fast method for detection of
ESBLs producing K.pneumoniae. as well as its useful in anabling the
microbiologist to forecast the out come of therapy and to develop
empirical guidance for therapy.
11
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