Detection Metallo-Beta-Lactamase Gene IMP-1 and IMP-2 of Pseudomonas aeruginosa Clinical Isolates In Sanglah Hospital Bali.
Detec%on Metallo‐Beta‐Lactamase Gene IMP‐1 and IMP‐2 of
Pseudomonas aeruginosa Clinical Isolates In Sanglah Hospital Bali
Ni Made Adi Tarini1,2*, Ni Nengah Dwi Fatmawati1,2,3, I Putu Bayu Mayura1
1Department
of Clinical Microbiology, Medical School, Faculty of Medicine, Udayana University
2Clinical Microbiology Laboratory, Sanglah General Hospital
3Molecular Biology Laboratory, Faculty of Medicine, Udayana University, Bali, Indonesia
Jl. PB. Sudirman, Denpasar, Bali, Indonesia
*nmatarini@unud.ac.id
BACKGROUND
RESULTS
Pseudomonas aeruginosa is a pathogen frequently found as an
agent of Hospital Acquired Infec7ons. This bacterium is very
easy to be resistant to several types of an7bio7cs through
various mechanisms. Carbapenem such as Imipenem and
Meropenem is a potensial op7on for the therapy of this
bacterium, but unfortunately P. aeruginosa has an ability in
hydrolyzing these an7bio7cs through enzyme metallo‐β‐
lactamases (MBLs). Recently, IMP and VIM, MBLs enzyme
group are reported common from various countries, but no
data is reported for these enzymes in Indonesia especially in
Bali. In fact, the resistant data of P. aeruginosa against
Carbapenem group an7bio7cs such as Meropenem and
Imipenem is quite high in Sanglah General Hospital in 2014,
which were 35% and 45%, respec7vely
Culture on MacConkey agar media
Colonies of P.
aeruginosa
16sRNA Uniplex PCR of P. aeruginosa
５
bp
208 bp
bp
OBJECTIVE
!
To detect IMP‐1 and IMP‐2 genes of MDR P. aeruginosa
isolates in Clinical Microbiology Laboratory, Sanglah General
Hospital, Denpasar Bali, which are phenotypicall resistant to
an7bio7cs, Imipenem and Meropenem using PCR.
M= marker 100 bp
IMP‐1 and IMP‐2 Gene Uniplex PCR
500 bp
MATERIALS and METHODS
587 bp
A total of 86 glycerol stock isolates of P. aeruginosa from
clinical samples
Culture
All isolates were cultured
on MacConkey Agar
Incubated aerobically at 35
± 2o C, 18 – 24 hour
Iden7fica7on and Drug
Suscep7bility Test by
VITEK‐2 based on CLSI
Standard
PCR
Bacterial genomic DNA was
isolated from colonies by
using Roche High PCR
Template Isola7on Kit (Roche
Life Science, Indianapolis,
USA
performed PCR with primers
16sRNA, blaIMP1 and bla
IMP2 for (Shibata et al),
annealing temperature at
55oC for IMP‐1 and 48 for
IMP‐2 and 16sRNA
Electrophoresis in
agarose gel 1.5 %
REFERENCES
• Arunagiri, K., Sekar, B., Sangeetha, G., John, J. (2012). Detec7on and
Characteriza7on of Metallo‐β‐Lactamases in Pseudomonas aeruginosa by
phenotypic and Molecular Methods from Clinical Samples in Ter7ary Care
Hospital, West Indian Med J, 61(8), 778‐783.
• Shibata, N., Doi, Y., Yamane, K., et al.(2003). PCR Typing of Gene7c
determinats for metallo‐β‐lactamases and integrases carried by gram‐
nega7ve bacteria isolated in Japan with focus on the class 3 integron. J
Clin Microbiol., 41, 5407‐5413.
• Sepehriseresht, S., Boroumand, M.,A., Pourgholi, L., et al. (2012).
Detec7on of VIM and IPM‐type Metallo‐β‐Lactamases in Pseudomonas
aeruginosa Clinical Isolates. Archives of Iranian Medicine, 15(11), 670‐673
!
100 bp
M = marker 100 bp
K‐1 = nega7ve control
68, 69,80 = posi7ve for IMP‐1 genes
There was no isolates posi%ve for IMP‐2 gene
CONCLUSION
All isolates were subjected to PCR for detec7on of IMP‐1 and
IMP‐2. The result showed that 9 isolates were posi7f IMP‐gene
(10.5%), but there was no isolates posi7ve for IMP‐2 gene. This
study showed the similar condi7on with the MBL gene research
studies from the other countries, especially for the gene IMP‐1 .
Detec7on and molecular characteriza7on of MBL‐producing P.
aeruginosa strains is very important for infec7on control
purposes. Currently, this study is s7ll con7nued for detec7on of
another MBL genes.
Acknowledgments
This work was financially supported by Hibah LITBANG Medicine Faculty
Udayana University, Bali, Indonesia 2014.
We thank Wahyu Hidaya7 (Molecular Biology Laboratory staff), Putu
Yuliandari, M.D. (Clinical Microbiology Laboratory, Faculty of Medicine staff),
and Ni Wayan Nilawa7 (Clinical Microbiology Laboratory Sanglah General
Hospital staff) for their technical supports.
Pseudomonas aeruginosa Clinical Isolates In Sanglah Hospital Bali
Ni Made Adi Tarini1,2*, Ni Nengah Dwi Fatmawati1,2,3, I Putu Bayu Mayura1
1Department
of Clinical Microbiology, Medical School, Faculty of Medicine, Udayana University
2Clinical Microbiology Laboratory, Sanglah General Hospital
3Molecular Biology Laboratory, Faculty of Medicine, Udayana University, Bali, Indonesia
Jl. PB. Sudirman, Denpasar, Bali, Indonesia
*nmatarini@unud.ac.id
BACKGROUND
RESULTS
Pseudomonas aeruginosa is a pathogen frequently found as an
agent of Hospital Acquired Infec7ons. This bacterium is very
easy to be resistant to several types of an7bio7cs through
various mechanisms. Carbapenem such as Imipenem and
Meropenem is a potensial op7on for the therapy of this
bacterium, but unfortunately P. aeruginosa has an ability in
hydrolyzing these an7bio7cs through enzyme metallo‐β‐
lactamases (MBLs). Recently, IMP and VIM, MBLs enzyme
group are reported common from various countries, but no
data is reported for these enzymes in Indonesia especially in
Bali. In fact, the resistant data of P. aeruginosa against
Carbapenem group an7bio7cs such as Meropenem and
Imipenem is quite high in Sanglah General Hospital in 2014,
which were 35% and 45%, respec7vely
Culture on MacConkey agar media
Colonies of P.
aeruginosa
16sRNA Uniplex PCR of P. aeruginosa
５
bp
208 bp
bp
OBJECTIVE
!
To detect IMP‐1 and IMP‐2 genes of MDR P. aeruginosa
isolates in Clinical Microbiology Laboratory, Sanglah General
Hospital, Denpasar Bali, which are phenotypicall resistant to
an7bio7cs, Imipenem and Meropenem using PCR.
M= marker 100 bp
IMP‐1 and IMP‐2 Gene Uniplex PCR
500 bp
MATERIALS and METHODS
587 bp
A total of 86 glycerol stock isolates of P. aeruginosa from
clinical samples
Culture
All isolates were cultured
on MacConkey Agar
Incubated aerobically at 35
± 2o C, 18 – 24 hour
Iden7fica7on and Drug
Suscep7bility Test by
VITEK‐2 based on CLSI
Standard
PCR
Bacterial genomic DNA was
isolated from colonies by
using Roche High PCR
Template Isola7on Kit (Roche
Life Science, Indianapolis,
USA
performed PCR with primers
16sRNA, blaIMP1 and bla
IMP2 for (Shibata et al),
annealing temperature at
55oC for IMP‐1 and 48 for
IMP‐2 and 16sRNA
Electrophoresis in
agarose gel 1.5 %
REFERENCES
• Arunagiri, K., Sekar, B., Sangeetha, G., John, J. (2012). Detec7on and
Characteriza7on of Metallo‐β‐Lactamases in Pseudomonas aeruginosa by
phenotypic and Molecular Methods from Clinical Samples in Ter7ary Care
Hospital, West Indian Med J, 61(8), 778‐783.
• Shibata, N., Doi, Y., Yamane, K., et al.(2003). PCR Typing of Gene7c
determinats for metallo‐β‐lactamases and integrases carried by gram‐
nega7ve bacteria isolated in Japan with focus on the class 3 integron. J
Clin Microbiol., 41, 5407‐5413.
• Sepehriseresht, S., Boroumand, M.,A., Pourgholi, L., et al. (2012).
Detec7on of VIM and IPM‐type Metallo‐β‐Lactamases in Pseudomonas
aeruginosa Clinical Isolates. Archives of Iranian Medicine, 15(11), 670‐673
!
100 bp
M = marker 100 bp
K‐1 = nega7ve control
68, 69,80 = posi7ve for IMP‐1 genes
There was no isolates posi%ve for IMP‐2 gene
CONCLUSION
All isolates were subjected to PCR for detec7on of IMP‐1 and
IMP‐2. The result showed that 9 isolates were posi7f IMP‐gene
(10.5%), but there was no isolates posi7ve for IMP‐2 gene. This
study showed the similar condi7on with the MBL gene research
studies from the other countries, especially for the gene IMP‐1 .
Detec7on and molecular characteriza7on of MBL‐producing P.
aeruginosa strains is very important for infec7on control
purposes. Currently, this study is s7ll con7nued for detec7on of
another MBL genes.
Acknowledgments
This work was financially supported by Hibah LITBANG Medicine Faculty
Udayana University, Bali, Indonesia 2014.
We thank Wahyu Hidaya7 (Molecular Biology Laboratory staff), Putu
Yuliandari, M.D. (Clinical Microbiology Laboratory, Faculty of Medicine staff),
and Ni Wayan Nilawa7 (Clinical Microbiology Laboratory Sanglah General
Hospital staff) for their technical supports.