Multiplex PCR for rapid detection of genes encoding Oxa and metallo-lactamases in Acinetobacter spp.

Abstract number: P1715

Mostachio A., van der Heijden I., Miranda L., Boszczowski I., Miranda L., Rossi F., Araújo E., Levin A., Costa S.

Acinetobacter spp, resistance to carbapenems, have become common in hospitals worldwide. Carbapenem resistance mechanisms described in A. baumannii include hydrolysis by b-lactamases, alterations in outer membrane proteins and penicillin-binding proteins, and increased activity of efflux pumps. However, carbapenemases, such as metallo-b-lactamase (MBL) or oxacillinases, are the most concerning.

Objective: The present study aimed to develop a multiplex PCR assay to detect and differentiate alleles coding oxacillinase and MBL in nosocomial carbapenem resistant Acinetobacter spp.

Material: The study included a total of sixty-eight isolates of A. baumannii, sixty four carbapenem-resistant and four carbapenem-susceptible strains. These isolates were obtained from 4 different hospitals of the state of São Paulo, Brazil. The following reference strains were used in this study: P. aeruginosa producing IMP-1, VIM-1, SIM-1 enzymes and A. baumannii producing Oxa-23 and Oxa-25 enzymes as positive controls and A. baumannii ATCC 19606 was used as a negative control. The isolates were identified by API (NE) (Biomerieux-France). Minimum inhibitory concentrations (MICs) of carbapenem were determined by broth microdilution and interpreted using Clinical Laboratory Standards Institute (CLSI) breakpoints. MBL ETEST was performed following the manufacturer's recommendations (AB Biodisk (North America Inc., N.J.) to confirm the MBL expression phenotypically in these strains.

Amplification by polymerase chain reaction with oligonucleotide primers specific for ISAba1 region was performed as described elsewhere

Results: Among, 64 carbapenem-resistant A. baumannii strains isolated from 4 Brazilian hospitals, blaoxa-23-like was present in 22 and IMP-1 only in 4 isolates. ISAba1 was identified in all carbapenem resistant strains except one. The imipenem MIC among blaoxa-23-like positive strains ranged from 16 to 128 ug/ml and was lower compared with IMP-1 positive strains (MIC > 128 ug/ml). Phenotypically expression of metallo-b-lactamase was confirmed by MBL-ETEST in all 4 IMP-1 positive strains.

Conclusion: The multiplex PCR results detected and distinguished alleles coding oxacillinase and MBL carbapenemases in carbapenem resistant A. baumannii and was consistent with the previous single PCR assays and could be an useful tool to understanding the dissemination of resistance of this microorganism in the hospital setting.

Session Details

Date: 16/05/2009
Time: 00:00-00:00
Session name: 19th European Congress of Clinical Microbiology and Infectious Diseases
Location: Helsinki, Finland, 16 - 19 May 2009
Presentation type:
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