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Successful multidrug-resistant clones are increasing in prevalence globally, which makes the ability to identify these clones urgent. However, adequate, easy-to-perform, and reproducible typing methods are lacking. We investigated whether DiversiLab (DL), an automated repetitive-sequence-based PCR bacterial typing system (bioMérieux), is suitable for comparing isolates analyzed at different geographic centers. A total of 39 Escherichia coli and 39 Klebsiella species isolates previously typed by the coordinating center were analyzed. Pulsed-field gel electrophoresis (PFGE) confirmed the presence of one cluster of 6 isolates, three clusters of 3 isolates, and three clusters of 2 isolates for each set of isolates. DL analysis was performed in 11 centers in six different countries using the same protocol. The DL profiles of 425 E. coli and 422 Klebsiella spp. were obtained. The DL system showed a lower discriminatory power for E. coli than did PFGE. The local DL data showed a low concordance, as indicated by the adjusted Rand and Wallace coefficients (0.132 to 0.740 and 0.070 to 1.0 [ E. coli ] and 0.091 to 0.864 and 0.056 to 1.0 [ Klebsiella spp.], respectively). The central analysis showed a significantly improved concordance (0.473 to 1.0 and 0.290 to 1.0 [ E. coli ] and 0.513 to 0.965 and 0.425 to 1.0 [ Klebsiella spp.], respectively). The misclassifications of profiles for individual isolates were mainly due to inconsistent amplification, which was most likely due to variations in the quality and amounts of the isolated DNA used for amplification. Despite local variations, the DL system has the potential to indicate the occurrence of clonal outbreaks in an international setting, provided there is strict adherence to standardized, reproducible DNA isolation methods and analysis protocols, all supported by a central database for profile comparisons.

Multidrug-resistant Acinetobacter baumannii (MDRAB) is an increasing problem worldwide. Prevalence of carbapenem resistance in Acinetobacter spp. due to acquired carbapenemase genes is not known in Finland. The purpose of this study was to examine prevalence and clonal spread of multiresistant A. baumannii group species, and their carbapenemase genes. A total of 55 Acinetobacter isolates were evaluated with repetitive PCR (DiversiLab) to analyse clonality of isolates, in conjunction with antimicrobial susceptibility profile for ampicillin/sulbactam, colistin, imipenem, meropenem, rifampicin and tigecycline. In addition, a new real-time PCR assay, detecting most clinically important carbapenemase genes just in two multiplex reactions, was developed. The assay detects genes for KPC, VIM, IMP, GES-1/-10, OXA-48, NDM, GIM-1, SPM-1, IMI/NMC-A, SME, CMY-10, SFC-1, SIM-1, OXA-23-like, OXA-24/40-like, OXA-58 and ISAbaI-OXA-51-like junction, and allows confident detection of isolates harbouring acquired carbapenemase genes. There was a time-dependent, clonal spread of multiresistant A. baumannii strongly correlating with carbapenamase gene profile, at least in this geographically restricted study material. The new carbapenemase screening assay was able to detect all the genes correctly suggesting it might be suitable for epidemiologic screening purposes in clinical laboratories.

The present study examined the suitability of matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for the rapid grouping of bacterial isolates, i.e. dereplication. Dereplication is important in large-scale isolation campaigns and screening programs since it can significantly reduce labor intensity, time and costs in further downstream analyses. Still, current dereplication techniques are time consuming and costly. MALDI-TOF MS is an attractive tool since it performs fast and cheap analyses with the potential of automation. However, its taxonomic resolution for a broad diversity of bacteria remains largely unknown. To verify the suitability of MALDI-TOF MS for dereplication, a total of 249 unidentified bacterial isolates retrieved from the rhizosphere of potato plants, were analyzed with both MALDI-TOF MS and repetitive element sequence based polymerase chain reaction (rep-PCR). The latter technique was used as a benchmark. Cluster analysis and inspection of the profiles showed that for 204 isolates (82%) the taxonomic resolution of both techniques was comparable, while for 45 isolates (18%) one of both techniques had a higher taxonomic resolution. Additionally, 16S rRNA gene sequence analysis was performed on all members of each delineated cluster to gain insight in the identity and sequence similarity between members in each cluster. MALDI-TOF MS proved to have higher reproducibility than rep-PCR and seemed to be more promising with respect to high-throughput analyses, automation, and time and cost efficiency. Its taxonomic resolution was situated at the species to strain level. The present study demonstrated that MALDI-TOF MS is a powerful tool for dereplication.