Carbapenem-resistant Enterobacter cloacae harbouring IMI2 gene in Finland

Abstract number: P1689

Koskela S., Antikainen J., Vaara M., Kirveskari J.

Objectives: Biochemical methods have been considered insufficient to identify the carbapenem resistance mechanisms. To supplement these gaps, a Real-Time multiplex PCR was developed for screening of clinically significant carbapenem resistance genes among Enterobacteriaceae, Acinetobacter sp. and Pseudomonas sp.

Methods: The Real-Time PCR assay design was divided in two multiplex reactions. The first multiplex was designed to detect VIM 1–22, IMP 1–24, OXA-48, KPC 1–7, GES 1–10, SPM, NMC-A, IMI 1–2, SME 1–3, GIM-1, and SIM-1 genes. The other multiplex was designed for OXA genes with carbapenemase properties only, including OXA-24, -25, 26, -40, -72, OXA-23, -27, OXA-50, OXA-51, 64–71, 75–78, 83–89, -92, -94, -95, OXA-55, OXA-58, OXA-60, and OXA-62. SYBR Green chemistry was chosen to allow numerous genes to be detected within single reaction. A melting curve analysis was used to preliminarily identify putative molecular mechanism of positive samples, which were confirmed with sequencing with reference primers. This assay was used to screen putative ESBL strains systematically. Moreover, the screening assay is part of routine diagnostics to screen putative Enterobacteriaceae species with reduced susceptibility (meropenem disk diameter <23 mm), or reduced MIC (I/R) to either meropenem or ertapenem.

Results: Over 250 putative ESBL strains were systemically screened to evaluate prevalence of carbapenemase producing strains, but they were not detected. In contrast, a highly carbapenem resistant Enterobacter cloacae strain harbouring IMI-2 gene was isolated from a patient in intensive care unit. The IMI-2 positive strain was highly resistant to meropenem (MIC > 32), imipenem (MIC > 32) and ertapenem (MIC > 256), but interestingly not, however, resistant to third generation cephalosporins, or trimetoprim/sulfa.

Conclusion: Large reservoir of carbapenem resistance genes in environmental species combined with increasing carbapenem use provoke the risk of emergence of rare or new carbapenemase genes, which may remain undetected even though broad combinations of biochemical and molecular techniques are implemented. Using the new high-throughput screening assay an E. cloacae harbouring IMI-2 gene was identified. To our knowledge, an E. cloacae with IMI-2 gene has previously been reported only once, in China. Furthermore, this is the first reported Finnish Enterobacteriaceae strain harbouring a carbapenemase gene as well.