Irrespective of the various advances of MALDI-TOF MS compared with biochemical methods, resolution of certain taxonomic groups still remain a daunting challenge. One of these difficult groups is the E. cloacae complex. Indeed, reference strains of E. cloacae itself and E. nimipressuralis were identified correctly and reliably using MALDI-TOF MS. In contrast, E. asburiae, E. hormaechei, E. kobei and E. ludwigii could not be

delineated from E. cloacae (Table 6). Eleven of 56 (20%) clinical isolates precharacterized as E. cloacae by biochemical GSK126 chemical structure methods could only be assigned to the E. cloacae complex and not to a certain species (Table 5). This is not satisfying with regard to a reproducible and reliable method for species delineation within the E. cloacae complex. Another method feasible for routine analysis with regard to time-efficiency and reliability are real-time PCRs. More recently, Pham et al. (2007) identified the gene of the molecular cochaperon DnaJ (dnaJ) as a gene with higher discriminatory power among Enterobacteriaceae than 16S rDNA, tuf and atpD genes. We generated alignments for the E. cloacae complex based on different genes like oriC, rpoB or gyrB. Again,

dnaJ turned out to be the most powerful marker for delineation of E. cloacae from the other species TGF-beta pathway of the complex. The selectivity of the primers and probe based on dnaj was tested both by homology searches of a nucleotide database (blastn) and by screening of seven E. cloacae and 56 non-E. cloacae strains including at least one representative of each species belonging to the E. cloacae complex. Neither false negatives nor false positives were recorded. The combination with ntb2 as IAC allowed exclusion

of the presence of inhibitory substances and dysfunctions of the PCR in case of dnaJ-negative results. Application of the duplex real-time PCR to clinical isolates, biochemically precharacterized as E. cloacae, resulted in the identification of 53 isolates as E. cloacae. Three isolates were identified as non-E. cloacae isolates. As MALDI-TOF MS identified these isolates as: (a) E. hormaechei (log score value 2.39) or E. cloacae (2.32); (b) E. kobei (2.24 ± 0.08) or E. cloacae (2.20 ± 0.07) and (c) E. asburiae (2.15 ± 0.08) or E. cloacae (2.14 ± 0.01), and E. cloacae Liothyronine Sodium was excluded by the duplex real-time PCR, we suggest that these isolates are indeed: (A) E. hormaechei; (B) E. kobei and (C) E. asburiae regarding the known difficulties of biochemical discrimination of these species. In conclusion, the duplex real-time PCR described here has high selectivity and is suitable for reliable identification of E. cloacae. Exclusive use of MALDI-TOF MS does not have the discriminatory power for clear and reliable identification of certain species within the E. cloacae complex. However, supplementing MALDI-TOF MS with determination of the presence or absence of E.