Ninety-six genetically diverse multidrug-resistant clinical isolates of Acinetobacter baumannii from 25 hospitals in 17 European countries were screened by PCR for specific carbapenemase-hydrolyzing class D ␤-lactamase (CHDL) genes and by PCR-based replicon typing for the presence of 19 different plasmid replicase (rep) gene homology groups (GRs). Results were confirmed by DNA sequencing where necessary. All 96 isolates contained at least 1 (with a maximum of 4) of the 19 groups of rep genes. Groups detected were GR6 (repAci6; 93 isolates), GR2 (including repAci1 [67 isolates] and repAci2 [3 isolates]), GR16 (repApAB49; 12 isolates), GR12 (p2ABSDF0001; 10 isolates), GR3 (repAci3; 4 isolates), GR4 (repAci4; 3 isolates), GR10 (repAciX; 1 isolate), and GR14 (repp4AYE; 1 isolate). Variations in rep gene content were observed even among epidemiologically related isolates. Genes encoding OXA-58-like CHDLs (22 isolates) were associated with carriage of the repAci1, repAci3, repAci4, and repAciX genes, genes encoding OXA-40-like CHDLs (6 isolates) were associated with repAci2 and p2ABSDF0001, and genes encoding OXA-23-like CHDLs (8 isolates) were associated with repAci1. Most intrinsic Acinetobacter plasmids are non-self-transferable, but the almost ubiquitous repAci6 gene was strongly associated with a potential tra locus that could serve as a general system for plasmid mobilization and consequent horizontal transmission of plasmids and their associated antibiotic resistance genes among strains of A. baumannii.

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In total, 96 carbapenem-resistant isolates of Acinetobacter baumannii were obtained from 25 hospitals in 17 European countries. Imipenem MICs ranged from <4 to 128 mg/L on retesting by Etest, with MICs > or =16 mg/L being associated with the carriage of genes encoding at least one other class D carbapenemase in addition to the intrinsic OXA-51-like enzyme. Molecular typing results obtained by random amplified polymorphic DNA analysis, followed by pulsed-field gel electrophoresis (PFGE) of ApaI-digested chromosomal DNA, were highly congruent, with 17 different PFGE types being delineated at a cut-off similarity level of 85%. With few exceptions, multiple isolates from a single hospital belonged to the same PFGE type. Seven sequence groups were identified among the 96 A. baumannii isolates, with the majority of isolates (n = 81) belonging to the previously defined sequence groups 1 and 2, which each included eight PFGE types. These two multinational lineages included the previously defined European clones II and I, respectively, but the problem of resistant A. baumannii in Europe appeared not to be confined solely to these two European clones. Rather, two broader lineages of carbapenem-resistant A. baumannii now seem to be spreading throughout Europe.

Aim-To develop and evaluate a TaqMan™ polymerase chain reaction (PCR) for the rapid identification and speciation of candida species. Methods-Species specific primer and probe sets were designed for the identification of Candida albicans, C parapsilosis, C tropicalis, C krusei, C kefyr, and C glabrata from clinical isolates in a 5' exonuclease (TaqMan™) assay. The probes were labelled with three fluorescent dyes to enable diVerentiation between species when three primer and probe sets were combined in two multiplexes. The specificity of these assays was evaluated against a range of National Collection of Pathogenic Fungi strains, clinical isolates of yeast, bacterial and viral pathogens. Results-The primer and probe sets have been shown to be 100% specific for their respective species; there was no crossreaction between any set and human DNA, or extracts from other candida species, fungal, bacterial, or viral pathogens tested. Extracts from two clinical isolates, originally identified as C albicans on the basis of germ tube formation, were not amplified by any of the primer and probe sets. These isolates have been putatively re-identified as C dubliniensis after sequencing of the variable internal transcribed spacer region ITS2 and lack of growth at 45°C. Conclusion-ThisTaqMan assay provides a rapid alternative to conventional culture based techniques for the identification and speciation of the most frequently isolated candida species. The simple extraction method followed by TaqMan PCR can identify the six species mentioned in four hours. (J Clin Pathol 2001;54:362-366)