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The emergence of Acinetobacter carbapenem resistance in the Czech Republic is associated with the spread of A. baumannii strains of EU clone II. The variation in susceptibility in these strains is likely to result from both the horizontal spread of resistance genes and differential expression of intrinsic genes.

In a recent study, a large proportion of multi-drug-resistant (MDR) Acinetobacter baumannii strains that were isolated from hospitalized patients in the Czech Republic was found to belong to two major groups (A and B). These groups appeared to be similar to epidemic clones I and II, respectively, which were identified previously among outbreak strains from north-western European hospitals. The aim of the present study was to assess in detail the genetic relatedness of Czech A. baumannii strains and those of epidemic clones I and II by using ribotyping with HindIII and HincII and by AFLP fingerprinting. The study collection included 70 MDR strains that were isolated in 30 Czech hospitals in 1991-2001, 15 susceptible Czech strains from 1991 to 1996 and 13 reference strains of clones I and II from 1982 to 1990. One major HindIII/HincIII ribotype (R1-1) was observed in 38 MDR Czech strains and eight reference strains of clone I, whereas another major ribotype (R2-2) was observed in 11 MDR Czech strains and in three reference strains of clone II. A selection of 59 Czech strains (representative of all ribotypes) and the 13 reference strains were investigated by AFLP fingerprinting. At a clustering level of 83 %, two large clusters could be distinguished: cluster 1 included all reference strains of clone I and 25 MDR Czech strains, whilst cluster 2 contained all reference strains of clone II and 11 MDR Czech strains. There was a clear correlation between the groupings by AFLP analysis and by ribotyping, as all strains with ribotype R1-1 and four strains with slightly different ribotypes were found in AFLP cluster 1, whereas all strains with ribotype R2-2 and seven strains with similar ribotypes were in AFLP cluster 2. Thus, 41 and 21 MDR Czech strains could be classified as belonging to clones I and II, respectively. The remaining eight MDR and 15 susceptible strains were highly heterogeneous and were distinct from clones I and II by both AFLP fingerprinting and ribotyping. These results indicate that the two predominant groups observed among MDR Czech A. baumannii strains from the 1990s are genetically congruent with the northwestern European epidemic clones that were found in the 1980s. Recognition of these clinically relevant, widespread clones is important in infection prevention and control; they are also interesting subjects to study genetic mechanisms that give rise to their antibiotic resistance and epidemic behaviour.

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Acinetobacter genospecies (genomic species) 10 and 11 were described by Bouvet and Grimont in 1986 on the basis of DNA–DNA reassociation studies and comprehensive phenotypic analysis. In the present study, the names Acinetobacter bereziniae sp. nov. and Acinetobacter guillouiae sp. nov., respectively, are proposed for these genomic species based on the congruence of results of polyphasic analysis of 33 strains (16 and 17 strains of genomic species 10 and 11, respectively). All strains were investigated by selective restriction fragment amplification (i.e. AFLP) analysis rpoB sequence analysis, amplified rDNA restriction analysis and tDNA intergenic length polymorphism analysis, and their nutritional and physiological properties were determined. Subsets of the strains were studied by 16S rRNA gene sequence analysis and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS or had been classified previously by DNA–DNA reassociation. Results indicate that A. bereziniae and A. guillouiae represent two phenetically and phylogenetically distinct groups within the genus Acinetobacter. Based on the comparative analysis of housekeeping genes (16S rRNA and rpoB genes), these species together represent a monophyletic branch within the genus. Despite their overall phenotypic similarity, the ability to oxidize d-glucose and to grow at 38 °C can be used in the presumptive differentiation of these two species from each other: with the exception of three strains that were positive for only one test, A. bereziniae strains were positive for both tests, whereas A. guillouiae strains were negative in these tests. The strains of A. bereziniae originated mainly from human clinical specimens, whereas A. guillouiae strains were isolated from different environmental sources in addition to human specimens. The type strain of A. bereziniae sp. nov. is LMG 1003T (=CIP 70.12T =ATCC 17924T) and that of A. guillouiae sp. nov. is LMG 988T (=CIP 63.46T =ATCC 11171T =CCUG 2491T).

The taxonomic status of seven glucose-non-acidifying, non-proteolytic Acinetobacter strains characterized by forming small colonies on agar media was studied. With one exception, all strains were from human specimens. They could be distinguished from all described Acinetobacter (genomic) species by their ability to grow on ethanol and acetate as sole sources of carbon but not on 22 other substrates tested including DL-lactate or DL-4-aminobutyrate. DNA-DNA hybridization studies, 16S rRNA gene sequence analysis, amplified rDNA restriction analysis and DNA polymorphism analysis by AFLP showed that these strains represent a hitherto unknown species of the genus Acinetobacter, for which the name Acinetobacter parvus (type strain LMG 21765 T =LUH 4616 T =NIPH 384 T =CCM 7030 T ) is proposed.The genus Acinetobacter comprises non-motile, strictly aerobic, oxidase-negative, Gram-negative bacteria that grow well on simple media. Twenty-four (genomic) species are currently recognized within the genus (Bouvet & Grimont, 1986;Tjernberg & Ursing, 1989;Bouvet & Jeanjean, 1989;Gerner-Smidt & Tjernberg, 1993;Vaneechoutte et al., 1999;Nemec et al., 2001) and strains of these species usually form colonies of 1?0-2?0 mm in diameter after 24 h incubation under optimum growth conditions (Bouvet & Grimont, 1986;Nemec et al., 2001). In a taxonomic study of Acinetobacter clinical isolates (Nemec et al., 2000), two strains were found which formed notably small colonies on routine agar media and could not be identified as any known (genomic) species. These strains were glucose-non-acidifying, non-proteolytic, did not utilize any of the 14 carbon sources of the identification scheme of Bouvet & Grimont (1987) and had highly similar amplified rDNA restriction analysis (ARDRA) profiles. Later, five strains similar to the two strains were found among archive strains in our collections. The aim of the present study was to define the taxonomic status of these strains by a polyphasic analysis.The seven strains used in this study are listed in Table 1. All had the properties of the genus Acinetobacter (Juni, 1984), i.e. they were Gram-negative, strictly aerobic, oxidasenegative, non-motile coccobacilli, and were positive in the transformation assay of Juni (1972). The methods for genotypic characterization included ARDRA, AFLP fingerprinting and comparative 16S rDNA sequence analysis. Phenotypic characterization was done essentially according to Bouvet & Grimont (1987) LMG 19082, which produced large amounts of exopolysaccharides, were subjected to a mild alkaline hydrolysis step before cell lysis, as described by Willems et al. (2001). The G+C content of the DNA was determined by HPLC according to the method of Mesbah et al. (1989). Nonmethylated phage l DNA (Sigma) was used as the calibration reference. DNA-DNA hybridizations were performed using a modification of the microplate method described by Ezaki et al. (1989) and Goris et al. (1998). Hybridizations were performed at 37 uC in a hybridization solution [26 SSC, 56Denhardt's solution, ...

Two outbreaks of multidrug-resistant Acinetobacter baumannii occurred in our hospital. The outbreak strains were eventually isolated from respiratory ventilators, an apparatus used to cool or warm patients, and four continuous veno-venous hemofiltration machines. Removing dust from the machines and replacing all dust filters brought the outbreaks to an end.

The AdeABC genes are common in A. baumannii, but may be absent in some, mostly fully susceptible strains. Decreased susceptibility to netilmicin (MIC 4-32 mg/L) is associated with both the presence of these genes and multidrug resistance and may be indicative of AdeABC overexpression.