Lenka Krizova

Genotypic and phenotypic characterization of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex with the proposal of Acinetobacter pittii sp. nov. (formerly Acinetobacter genomic species 3) and Acinetobacter nosocomialis sp. nov. (formerly Acinetobacter genomic species 13TU).

Acinetobacter genomic species (gen. sp.) 3 and gen. sp. 13TU are increasingly recognized as clinically important taxa within the Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) complex. To define the taxonomic position of these genomic species, we investigated 80 strains representing the known diversity of the ACB complex. All strains were characterized by AFLP analysis, amplified rDNA restriction analysis and nutritional or physiological testing, while selected strains were studied by 16S rRNA and rpoB gene sequence analysis, multilocus sequence analysis and whole-genome comparison. Results supported the genomic distinctness and monophyly of the individual species of the ACB complex. Despite the high phenotypic similarity among these species, some degree of differentiation between them could be made on the basis of growth at different temperatures and of assimilation of malonate, l-tartrate levulinate or citraconate. Considering the medical relevance of gen. sp. 3 and gen. sp. 13TU, we propose the formal names Acinetobacter pittii sp. nov. and Acinetobacter nosocomialis sp. nov. for these taxa, respectively. The type strain of A. pittii sp. nov. is LMG 1035(T) (=CIP 70.29(T)) and that of A. nosocomialis sp. nov. is LMG 10619(T) (=CCM 7791(T)).

The Genomic Diversification of the Whole Acinetobacter Genus: Origins, Mechanisms, and Consequences

Bacterial genomics has greatly expanded our understanding of microdiversification patterns within a species, but analyses at higher taxonomical levels are necessary to understand and predict the independent rise of pathogens in a genus. We have sampled, sequenced, and assessed the diversity of genomes of validly named and tentative species of the Acinetobacter genus, a clade including major nosocomial pathogens and biotechnologically important species. We inferred a robust global phylogeny and delimited several new putative species. The genus is very ancient and extremely diverse: Genomes of highly divergent species share more orthologs than certain strains within a species. We systematically characterized elements and mechanisms driving genome diversification, such as conjugative elements, insertion sequences, and natural transformation. We found many error-prone polymerases that may play a role in resistance to toxins, antibiotics, and in the generation of genetic variation. Surprisingly, temperate phages, poorly studied in Acinetobacter, were found to account for a significant fraction of most genomes. Accordingly, many genomes encode clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems with some of the largest CRISPR-arrays found so far in bacteria. Integrons are strongly overrepresented in Acinetobacter baumannii, which correlates with its frequent resistance to antibiotics. Our data suggest that A. baumannii arose from an ancient population bottleneck followed by population expansion under strong purifying selection. The outstanding diversification of the species occurred largely by horizontal transfer, including some allelic recombination, at specific hotspots preferentially located close to the replication terminus. Our work sets a quantitative basis to understand the diversification of Acinetobacter into emerging resistant and versatile pathogens.

Multidrug-resistant epidemic clones among bloodstream isolates of Pseudomonas aeruginosa in the Czech Republic.

To determine whether the high proportion of antimicrobial resistance among hospital isolates of Pseudomonas aeruginosa in the Czech Republic is associated with the spread of multidrug-resistant clones, we investigated 108 bloodstream isolates collected prospectively in 2007. The isolates originated from 48 hospitals in 36 cities and were serotyped, tested for susceptibility to 10 anti-Pseudomonas agents and studied by multilocus sequence typing, macrorestriction analysis and class 1 integron typing. Forty-five isolates were fully susceptible, while 14 and 49 isolates were resistant to 1-2 and 3-9 agents, respectively. A total of 42 multilocus sequence types (ST) were identified, of which ST235 (serotype O11), ST175 (O4) and ST132 (O6) included 19, 16 and 5 isolates, respectively. These three STs encompassed 40 (82%) of 49 isolates resistant to more than two agents and originated from 29 hospitals in 22 cities. Isolates of the same ST had highly similar macrorestriction patterns. Twelve ST235 isolates harbored an integron variable region with the gene cassette array of aacA7-aadA6-orfD, while 15 ST175 isolates shared a region with the aadB-aadA13 array and all ST132 isolates carried a region with aacA4. A carbapenemase-encoding gene (bla(IMP-7)) was detected in a single strain (ST357). In conclusion, the multidrug resistance of Czech P. aeruginosa bloodstream isolates in 2007 was predominantly associated with three epidemic clones, one of which belongs to international clonal complex CC235.

Diversity and Evolution of AbaR Genomic Resistance Islands in Acinetobacter baumannii Strains of European Clone I

ABSTRACT To assess the diversity of AbaR genomic resistance islands in Acinetobacter baumannii European clone I (MLST clonal complex 1), we investigated 26 multidrug-resistant strains of this major clone isolated from hospitals in 21 cities of 10 European countries between 1984 and 2005. Each strain harbored an AbaR structure integrated at the same position in the chromosomal ATPase gene. AbaR3, including four subtypes based on variations in class 1 integron cassettes, and AbaR10 were found in 15 and 2 strains, respectively, whereas a new, unique AbaR variant was discovered in each of the other 9 strains. These new variants, designated AbaR11 to AbaR19 (19.8 kb to 57.5 kb), seem to be truncated derivatives of AbaR3, likely resulting from the deletions of its internal parts mediated by either IS26 elements (AbaR12 to AbaR19) or homologous recombination (AbaR11). AbaR3 was detected in all 10 strains isolated in 1984 to 1991, while AbaR11 to AbaR19 were carried only by strains isolated since 1997. Our results and those from previous publications suggest that AbaR3 is the original form of AbaR in European clone I, which may have provided strains of the lineage with a selective advantage facilitating their spread in European hospitals in the 1980s or before.

Identification of 50 Class D β-Lactamases and 65 Acinetobacter-Derived Cephalosporinases in Acinetobacter spp.

ABSTRACT Whole-genome sequencing of a collection of 103 Acinetobacter strains belonging to 22 validly named species and another 16 putative species allowed detection of genes for 50 new class D β-lactamases and 65 new Acinetobacter-derived cephalosporinases (ADC). All oxacillinases (OXA) contained the three typical motifs of class D β-lactamases, STFK, (F/Y)GN, and K(S/T)G. The phylogenetic tree drawn from the OXA sequences led to an increase in the number of OXA groups from 7 to 18. The topologies of the OXA and RpoB phylogenetic trees were similar, supporting the ancient acquisition of blaOXA genes by Acinetobacter species. The class D β-lactamase genes appeared to be intrinsic to several species, such as Acinetobacter baumannii, Acinetobacter pittii, Acinetobacter calcoaceticus, and Acinetobacter lwoffii. Neither blaOXA-40/143- nor blaOXA-58-like genes were detected, and their origin remains therefore unknown. The phylogenetic tree analysis based on the alignment of the sequences deduced from blaADC revealed five main clusters, one containing ADC belonging to species closely related to A. baumannii and the others composed of cephalosporinases from the remaining species. No indication of blaOXA or blaADC transfer was observed between distantly related species, except for blaOXA-279, possibly transferred from Acinetobacter genomic species 6 to Acinetobacter parvus. Analysis of β-lactam susceptibility of seven strains harboring new oxacillinases and cloning of the corresponding genes in Escherichia coli and in a susceptible A. baumannii strain indicated very weak hydrolysis of carbapenems. Overall, this study reveals a large pool of β-lactamases in different Acinetobacter spp., potentially transferable to pathogenic strains of the genus.

Acinetobacter bohemicus sp nov widespread in natural soil and water ecosystems in the Czech Republic

We investigated the taxonomic status of a phenetically unique group of 25 Acinetobacter strains which were isolated from multiple soil and water samples collected in natural ecosystems in the Czech Republic. Based on the comparative sequence analyses of the rpoB, gyrB, and 16S rRNA genes, the strains formed a coherent and well separated branch within the genus Acinetobacter. The genomic uniqueness of the group at the species level was supported by the low average nucleotide identity values (≤77.37%) between the whole genome sequences of strain ANC 3994(T) (NCBI accession no. APOH00000000) and the representatives of the known Acinetobacter species. Moreover, all 25 strains created a tight cluster clearly separated from all hitherto described species based on whole-cell protein profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and shared a unique combination of metabolic and physiological properties. The capacity to assimilate l-histidine and the inability to grow at 35°C differentiated them from their phenotypically closest neighbor, Acinetobacter johnsonii. We conclude that the 25 strains represent a novel Acinetobacter species, for which the name Acinetobacter bohemicus sp. nov. is proposed. The type strain of A. bohemicus is ANC 3994(T) (=CIP 110496(T)=CCUG 63842(T)=CCM 8462(T)).

A 63 kb genomic resistance island found in a multidrug-resistant Acinetobacter baumannii isolate of European clone I from 1977

OBJECTIVES Multidrug-resistant Acinetobacter strain HK302 was isolated from an outbreak of nosocomial infections in Switzerland in 1977. The aim of the present study was to assess whether this archive strain belongs to one of the known international clonal lineages of Acinetobacter baumannii and whether it harbours a genomic structure related to the AbaR1-like resistance islands. METHODS Multilocus sequence typing (MLST) and HindIII ribotyping were used to determine the taxonomic position of HK302 at the species and subspecies (clonal) levels. The position and structure of the putative resistance island were investigated by AbaR1-based PCR mapping followed by restriction analysis and partial sequencing of amplicons. A. baumannii AYE harbouring AbaR1 was used as a positive control for PCR mapping. RESULTS The MLST allelic profile (1-1-1-1-5-1-1) and HindIII ribotype of HK302 were typical of A. baumannii European (EU) clone I. In addition, an AbaR1-related region inserted into the ATPase gene at the same position as AbaR1 was found in HK302. PCR mapping and partial sequencing revealed that this region is structurally congruent with AbaR3, a 63.4 kb island described in an A. baumannii isolate from 2004. CONCLUSIONS A. baumannii HK302 belongs to EU clone I and harbours an AbaR3-like island related to resistance islands described in EU clone I strains. Our findings suggest that variants of these sophisticated genomic structures already existed in A. baumannii in the late 1970s.

TEM-1 β-lactamase as a source of resistance to sulbactam in clinical strains of Acinetobacter baumannii

OBJECTIVES Sulbactam is well known to have clinically relevant intrinsic activity against Acinetobacter baumannii. Although secondary resistance to this drug has long been reported in acinetobacters, virtually nothing is known about its molecular basis. The aim of this study was to test the hypothesis that β-lactamase TEM-1 is responsible for sulbactam resistance in A. baumannii. METHODS Seventeen clinical strains of A. baumannii were selected to represent different combinations of quantitative susceptibilities to sulbactam and molecular typing characteristics. The strains were screened by PCR for the presence of the blaTEM-1 gene and its variants. Amplicons encompassing the blaTEM genes, including their promoters, were sequenced. The expression and copy number of the blaTEM genes were assessed using semi-quantitative real-time PCR. Transfer of the blaTEM-1 gene into a susceptible A. baumannii strain was achieved by electroporation. RESULTS Six strains were negative for the blaTEM gene and had sulbactam MICs of 0.5-1.0 mg/L, 10 strains harboured blaTEM-1 and showed MICs ≥ 8.0 mg/L, except for one strain with an MIC of 2 mg/L, while the remaining strain carried blaTEM-19 and had an MIC of 1 mg/L. The level of blaTEM-1 expression positively correlated with the MICs of sulbactam (r = 0.92). Promoter P4 was linked to the blaTEM gene in all strains except for a P3-carrying strain (an MIC of 2 mg/L). Transformation of the susceptible A. baumannii strain with blaTEM-1 resulted in a 64-fold increase in sulbactam MIC and in resistance to ticarcillin and piperacillin, but no change in susceptibility to broad-spectrum generation cephalosporins, aztreonam or carbapenems. CONCLUSIONS The results presented suggest that TEM-1 represents a clinically relevant mechanism of sulbactam resistance in A. baumannii.

Acinetobacter variabilis sp. nov. (formerly DNA group 15 sensu Tjernberg & Ursing), isolated from humans and animals

We aimed to define the taxonomic status of 16 strains which were phenetically congruent with Acinetobacter DNA group 15 described by Tjernberg & Ursing in 1989. The strains were isolated from a variety of human and animal specimens in geographically distant places over the last three decades. Taxonomic analysis was based on an Acinetobacter-targeted, genus-wide approach that included the comparative sequence analysis of housekeeping, protein-coding genes, whole-cell profiling based on matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), an array of in-house physiological and metabolic tests, and whole-genome comparative analysis. Based on analyses of the rpoB and gyrB genes, the 16 strains formed respective, strongly supported clusters clearly separated from the other species of the genus Acinetobacter. The distinctness of the group at the species level was indicated by average nucleotide identity values of ≤82 % between the whole genome sequences of two of the 16 strains (NIPH 2171(T) and NIPH 899) and those of the known species. In addition, the coherence of the group was also supported by MALDI-TOF MS. All 16 strains were non-haemolytic and non-gelatinase-producing, grown at 41 °C and utilized a rather limited number of carbon sources. Virtually every strain displayed a unique combination of metabolic and physiological features. We conclude that the 16 strains represent a distinct species of the genus Acinetobacter, for which the name Acinetobacter variabilis sp. nov. is proposed to reflect its marked phenotypic heterogeneity. The type strain is NIPH 2171(T) ( = CIP 110486(T) = CCUG 26390(T) = CCM 8555(T)).

Characterization of a multidrug-resistant Acinetobacter baumannii strain carrying the blaNDM-1 and blaOXA-23 carbapenemase genes from the Czech Republic

Sir, Since its discovery in 2008, the metallo-b-lactamase (MBL) NDM-1 has been identified in different Enterobacteriaceae species and recently also in a number of other bacterial species isolated from water supplies in India, such as Vibrio cholerae and Pseudomonas spp. Genes encoding NDM b-lactamases have been additionally identified in multidrugresistant Acinetobacter spp. Here, we report on the identification and characterization of an Acinetobacter baumannii strain carrying the blaNDM-1 gene that was isolated in the Czech Republic in 2011. A. baumannii strain ANC 4097 was isolated in mid-2011 during a prospective, nationwide study in the Czech Republic focused on the Acinetobacter population structure and resistance mechanisms. It was recovered from the sputum of an elderly patient hospitalized in an intensive care unit (ICU) of a hospital located in the north-western part of the Czech Republic. The patient, who had a malignant tumour, died of complications of the underlying disease several days after the isolation of the strain. The MICs of b-lactams for ANC 4097 determined using Etest (bioMérieux, Solna, Sweden) showed high-level resistance to all b-lactams, including carbapenems (MICs of both imipenem and meropenem .32 mg/L) and broad-spectrum cephalosporins (MICs of both ceftazidime and cefepime .256 mg/L), as well as penicillins in combination with inhibitors (MICs of ampicillin/sulbactam and piperacillin/tazobactam .256 mg/L). The strain was also resistant to fluoroquinolones (MIC of ciprofloxacin .32 mg/L), aminoglycosides (MIC of amikacin .64 mg/L and MIC of gentamicin 32 mg/L) and tetracycline (MIC 128 mg/L). An MBL Etest (bioMérieux) revealed a 24-fold reduction of imipenem MIC when combined with EDTA, which suggested production of an MBL. Isolate ANC 4097 remained susceptible only to tigecycline (MIC 2 mg/L), doxycycline (MIC 2 mg/L), netilmicin (MIC 4 mg/L), tobramycin (MIC 0.5 mg/L) and colistin (MIC 0.25 mg/L). Strain ANC 4097 was genotyped by multilocus sequence typing (MLST) using the Pasteur Institute scheme (http://www.pasteur.fr/ recherche/genopole/PF8/mlst/). It belongs to sequence type (ST) 1 (allelic profile 1-1-1-1-5-1-1), which is typical of the European clone I epidemic lineage. PCR screening followed by sequencing of resistance determinants revealed that the strain harboured genes encoding five b-lactamases: NDM-1, class D carbapenemases OXA-23 (acquired) and OXA-69 (intrinsic), acquired narrowspectrum class A b-lactamase TEM-1 and intrinsic AmpC-type b-lactamase ADC. This strain co-harboured the tetA(A) tetracycline resistance gene and the aacC1, aphA1, aphA6 and aadA1 aminoglycoside resistance genes, but no armA 16S RNA methylase gene was identified. Insertion sequence ISAba1 was detected upstream of each of blaOXA-23, blaOXA-69 and blaADC, thus providing strong promoter sequences likely to enhance the expression of these genes in ANC 4097. In addition, the comM gene (encoding a polypeptide containing an ATPase domain) was found to be disrupted, which, together with the presence of the blaTEM-1, aacC1, aphA1, aadA1 and tetA(A) genes, suggested the presence of an AbaR3-like genomic resistance island typical of the European clone I lineage. Genetic structures associated with the blaOXA-23 and blaNDM-1 genes were analysed using PCR mapping and sequencing as described elsewhere. The blaNDM-1 gene was located in the previously characterized composite transposon Tn125 (two copies of ISAba125 bracketing a 7925 bp region), while the blaOXA-23 gene was found to be part of transposon Tn2008. In order to determine the genetic location of the blaOXA-23 and blaNDM-1 genes, plasmid DNA of ANC 4097 was isolated as described previously. Transfer of the ticarcillin resistance marker was attempted by both electroporation of the ANC 4097 plasmid suspension into A. baumannii BM4547 and liquid mating-out assays of the ANC 4097 and BM4547 strains at 378C. Selection was performed on agar plates supplemented with ticarcillin (100 mg/L). Transformants or transconjugants harbouring the blaNDM-1 and blaOXA-23 genes were not obtained, suggesting a chromosomal location of both genes, as described previously. This study reports on the first blaNDM-1-positive A. baumannii strain in the Czech Republic and adds to the body of evidence of the current spread of multidrug-resistant Acinetobacter harbouring this MBL in Europe.