Characterisation of carbapenem-resistance in carbapenemase-negative Klebsiella pneumoniae for development of an antibody-based rapid detection kit

Background

Carbapenem-resistant Klebsiella pneumoniae (Kp) is of serious concern in hospitals. Carbapenem-resistance is mediated by carbapenemases, however, for carbapenemase-negative carbapenem-resistant Kp (CNCRKp), a combination of ESBL and porin loss is an alternative resistance mechanism. Detection of carbapenemases by lateral flow assays is well-established, yet for CNCRKp no such test exists. We therefore sought to develop a rapid diagnostic test to identify CNCRKp.

Materials

Thirty-two CNCRKp clinical isolates from Germany were sequenced (MiSeq) and resistance to ertapenem, meropenem and imipenem was confirmed by agar dilution. For gene expression analysis, total RNA was extracted and qRT-PCR was performed with primers targeting ESBLs (SHV, CTX-M, TEM, DHA, OXA-1 and OXA-9) and porins (OmpK35 and OmpK36).

Results

All isolates were resistant to ertapenem, 25 (78 %) were resistant to meropenem and 17 (53 %) to imipenem according to EUCAST guidelines. Sequence analysis revealed deleterious mutations in the ompK35 and ompK36 genes in 14 and 26 isolates, respectively. All remaining isolates with intact gene sequences showed decreased OmpK35 and OmpK36 expression on mRNA level compared to the reference carbapenem-susceptible strain ATCC13883. Porin loss/reduced expression combined with SHV expression was associated only with ertapenem resistance. qRT-PCR data demonstrate that isolates expressing either DHA or an increased expression of CTX-M, OXA-1 or OXA-9 were resistant to all tested carbapenems. ESBL and porin genes were cloned into E. coli to retrieve recombinant proteins used for the generation of monoclonal antibodies (mAbs) by hybridoma technology. Expression of endogenous ESBLs and porins in lysates of Kp clinical isolates was detected in Western blot using our antigen-specific mAbs.

Conclusion

These data support the hypothesis that porin loss (deleted/mutated gene) or decreased expression in combination with overexpression of ESBLs is associated with carbapenem resistance in CNCRKp. Our results have direct impact on the combination of ESBL-/porin-specific mAbs in an immunochromatographic lateral flow assay to rapidly detect CNCRKp.