comparison of phenotypic methods and pcr for the detection

Comparison of phenotypic methods and PCR for the detection of

carbapenemase production in clinical Klebsiella pneumoniae isolates

Tekintas Y*1, Cilli FF2, Erac B3, Yasar M2, Aydemir SS2, Hosgor-Limoncu M 3 1Izmir Katip Celebi University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, Izmir,Turkey

2Ege University, Faculty of Medicine, Department of Medical Microbiology, Izmir, Turkey 3Ege University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, Izmir, Turkey

*E-mail: yamactekintas@yahoo.com

BACKGROUND

Multidrug resistant Klebsiella pneumoniae isolates are a major problem both in our country and the World.

Carbapenems are frequently used in the treatment of nosocomial K. pneumoniae infections. Nowadays, OXA and

Metallo beta lactamase (MBL) group enzymes are detected in the carbapenem resistant Klebsiella isolates. The

aim of this study is to compare phenotypic and genotypic methods for the detection of OXA-48 and MBLs, which

are thought to be responsible for carbapenem resistance of K. pneumoniae strains.

MATERIAL/METHODS

Carbapenemase presence and types were investigated in carbapenem resistant 54 K. pneumoniae strains isolated

from the Ege University Hospital Bacteriology Laboratory of Medical Microbiology Department. VITEK MS

and VITEK 2 Compact® automated systems were used for identification and antibiotic susceptibilities of the

strains, respectively. In carbapenem resistant strains, minimum inhibitor concentration (MIC) values of

meropenem were determined by gradient test method in the direction of EUCAST recommendations.

Phenotypic enzyme typing was done with the '' MASTDISCS ™ ID carbapenemase (Enterobacteriaceae)

detection disc set ''. The presence of OXA-48 and MBL (IMP, VIM, SIM, NDM) genes in the isolates was

investigated by polymerase chain reaction (PCR). Carbapenem Inactivation Method (CIM) was also used to

detect carbapenemase activity.

RESULTS

According to the VITEK 2 Compact® and gradient test results, 54 K. pneumoniae isolates were found to be

resistant to at least one carbapenem. Imipenem, meropenem and ertapenem MIC50 and MIC90 values were

determined as 32 μg / ml. Only OXA-48 was found in 33 of the strains and only NDM gene was detected in

two strains, but 19 strains were found to contain both genes by PCR. SIM, VIM, IMP genes were not

encountered in any of the isolates.

Table. Compatibility of phenotypic methods with PCR

CONCLUSIONS

MBL and OXA-48 genes were detected at high levels in carbapenem resistant K. pneumoniae strains isolated in

our hospital. Our results suggested that MASTDISC is highly compatible with PCR for single carbapenemase

gene harboring isolates, For isolates carrying two carbapenemase genes (OXA-48+NDM), MASTDISC should be

verified with PCR for understanding type of the enzyme. CIM has showed a low detection level in isolates only

containing OXA-48 (21%). The success rate is much higher in strains which carries NDM and OXA-48 genes

together (89%).

MASTDISC CIM

Carbapenemase Phenotype Number of strains Phenotype Number of strains

gene

blaOXA-48 n=33 MBL 0 (+) 7 (21%)

OXA-48 33 (100%) (-) 26 (79%)

blaOXA-48+blaNDM n=19 MBL 18 (95%) (+) 17 (89%)

OXA-48 1 (5%) (-) 2 (11%)

blaNDM n=2 MBL 2 (100%) (+) 1 (50%)

OXA-48 0 (-) 1 (50%)

MBL: Metallo Beta Lactamase, CIM: Carpabenemase Inactivation Method