Alexandr Nemec

We previously reported the presence of an OXA-23 carbapenemase in an undescribed species of the genus Acinetobacter isolated from horse dung at the Faculty of Veterinary Medicine, Ghent University, Belgium. Here we include six strains to corroborate the delineation of this taxon by phenotypic characterization, DNA-DNA hybridization, 16S rRNA gene and rpoB sequence analysis, % G+C determination, MALDI-TOF MS and fatty acid analysis. The nearly complete 16S rRNA gene sequence of strain UG 60467(T) showed the highest similarities with those of the type strains of Acinetobacter bouvetii (98.4 %), Acinetobacter haemolyticus (97.7 %), and Acinetobacter schindleri (97.2 %). The partial rpoB sequence of strain UG 60467(T) showed the highest similarities with 'Acinetobacter bohemicus' ANC 3994 (88.6 %), A. bouvetii NIPH 2281 (88.6 %) and A. schindleri CIP 107287T (87.3 %). Whole-cell MALDI-TOF MS analyses supported the distinctness of the group at the protein level. The predominant fatty acids of strain UG 60467(T) were C12 : 0 3-OH, C12 : 0, C16 : 0, C18 : 1ω9c and summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH). Strains UG 60467(T) and UG 60716 showed a DNA-DNA relatedness of 84 % with each other and a DNA-DNA relatedness with A. schindleri LMG 19576(T) of 17 % and 20 %, respectively. The DNA G+C content of strain UG 60467(T) was 39.6 mol%. The name Acinetobacter gandensis sp. nov. is proposed for the novel taxon. The type strain is UG 60467(T) ( = ANC 4275(T) = LMG 27960(T) = DSM 28097(T)).

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)).

This study aimed to define the taxonomic status of a phenetically distinct group of 16 strains which corresponds to Acinetobacter genomic species 'Close to 13TU', a provisional genomic species of the Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) complex recognized by Gerner-Smidt & Tjernberg in 1993. These strains have been isolated in different countries since the early 1990s and were mostly recovered from human clinical specimens. They were compared with 45 reference strains representing the known taxa of the ACB complex using taxonomic methods relevant to the genus Acinetobacter. Based on the sequence analysis of the concatenated partial sequences (2976 bp) of seven housekeeping genes, the 16 strains formed a tight and well supported cluster (intracluster sequence identity of ≥98.4%) clearly separated from the other members of the ACB complex (≤94.7%). The species status of the group was supported by average nucleotide identity values of ≤91.7% between the whole genome sequence of NIPH 973T (NCBI accession no. APOO00000000) and those of the other species. In addition, whole-cell MALDI-TOF MS analyses indicated the distinctness of the group at the protein level. Metabolic and physiological tests revealed several typical features of the group although they did not allow its reliable differentiation from the other members of the ACB complex. We conclude that the 16 strains represent a distinct species for which we propose the name Acinetobacter seifertii sp. nov. The type strain is NIPH 973T (= CIP 110471T = CCUG 34785T = CCM 8535T).

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)).

The amikacin resistance gene aphA6 was first detected in the nosocomial pathogen Acinetobacter baumannii and subsequently in other genera. Analysis of 133 whole-genome sequences covering the taxonomic diversity of Acinetobacter spp. detected aphA6 in the chromosome of 2 isolates of A. guillouiae, which is an environmental species, 1 of 8 A. parvus isolates, and 5 of 34 A. baumannii isolates. The gene was also present in 29 out of 36 A. guillouiae isolates screened by PCR, indicating that it is ancestral to this species. The Pnative promoter for aphA6 in A. guillouiae and A. parvus was replaced in A. baumannii by PaphA6, which was generated by use of the insertion sequence ISAba125, which brought a -35 sequence. Study of promoter strength in Escherichia coli and A. baumannii indicated that PaphA6 was four times more potent than Pnative. There was a good correlation between aminoglycoside MICs and aphA6 transcription in A. guillouiae isolates that remained susceptible to amikacin. The...

MALDI-TOF MS is currently becoming the method of choice for rapid identification of bacterial species in routine diagnostics. Yet, this method suffers from the inability to differentiate reliably between some closely related bacterial species including those of the Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) complex, namely A. baumannii and Acinetobacter nosocomialis. In the present study, we evaluated a protocol which was different from that used in the Bruker Daltonics identification system (MALDI BioTyper) to improve species identification using a taxonomically precisely defined set of 105 strains representing the four validly named species of the ACB complex. The novel protocol is based on the change in matrix composition from alpha-cyano-4-hydroxycinnamic acid (saturated solution in water:acetonitrile:trifluoroacetic acid, 47.5:50:2.5, v/v) to ferulic acid (12.5mgml(-1) solution in water:acetonitrile:formic acid 50:33:17, v/v), while the other steps of sample processing remain unchanged. Compared to the standard protocol, the novel one extended the range of detected compounds towards higher molecular weight, produced signals with better mass resolution, and allowed the detection of species-specific signals. As a result, differentiation of A. nosocomialis and A. baumannii strains by cluster analysis was improved and 13 A. nosocomialis strains, assigned erroneously or ambiguously by using the standard protocol, were correctly identified.

Page 1. CHAPTER 3 Nemec A, Dolzani L, Brisse S, van den Broek P, Dijkshoorn L. Diversity of aminoglycoside resistance genes and their association with class 1 integrons among strains of pan-European Acinetobacter baumannii clones. J Med Microbiol 2004; 53: 1233-1240. ...

... J Infect Dis 1983;148:346-357. • Seifert H, Gerner-Smidt P. Comparison of ribotyping and pulsed-field electrophoresis for mo-lecular typing of Acinetobacter isolates. J Clin Microbiol 1995;33:1402-1407. ... 15. Nemec A, Haywood-Farmer A, Mackie GA. ...

ABSTRACT Acinetobacter baumannii is a major nosocomial pathogen which typically affects critically ill patients in intensive care units. This organism is notorious for its resistance to antibiotics, its ability to spread epidemically, and its persistence in the hospital environment. Various genotypic methods have been developed to investigate the epidemiology of the organism at the strain level. Use of these meth- ods has shown that particular clones are mainly responsible for outbreaks and spread in hospitals. With the emergence of carbapenem resistance, options for treatment of A. baumannii infections are limited.

A collection of 95 strains of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex, isolated between 1991 and 1993 in the Prague Burn Center (BC), was studied. Ninety-one strains were isolated from 43 patients: 50 of them from burnt sites, 22 from endotracheal tube, 13 from urine, 3 from blood and 3 from venous catheter, and 4 strains were isolated from the hospital environment and the nursing staff. The strains were classified by restriction endonuclease fingerprinting of total DNA, plasmid profile analysis, ribotyping, comparison of antibiograms, biotyping and according to epidemiological data, into 31 relatedness groups each of them including 1 to 29 strains, likely to be isolates of the same strain. None of the methods used enabled to distinguish all groups. The importance of the polyphasic approach is emphasized since three multiresistant strains, isolated almost simultaneously in the BC, needed at least two methods to be distinguished (e.g. ribotyping and biotyping). Twenty-eight representative strains of different groups were identified by ribotyping: 18 of them were allocated to genomospecies 2 (A. baumannii), 5 to genomospecies 3 and 5 to genomospecies 13 sensu Tjernberg and Ursing. Only A. baumannii was found to spread among patients. Strains of two multiresistant groups persisted in the BC throughout the period studied and strains of one of these groups were responsible for an outbreak in the autumn of 1993. The methods mentioned above were used to describe 12 multiresistant strains isolated in three hospital wards in other localities. When ribotyped these strains were identified as A. baumannii. The strains of the same origin were identical in their typing profiles while the strains of different origins were easy to differentiate using any of the above methods; nevertheless, 2 of these groups were almost identical to 2 groups of multiresistant strains isolated in the BC.

To assess whether the high rate of antimicrobial resistance among bloodstream isolates of Pseudomonas aeruginosa in the Czech Republic is associated with the clonal spread of multidrug resistant (MDR) strains. A total of 30 bloodstream isolates obtained in 2007 were studied, including 20 MDR isolates (from 20 hospitals in 15 cities) and 10 susceptible control isolates. Each of the MDR isolates was resistant to at least three of the following agents: piperacillin, ceftazidime, meropenem, ciprofloxacin, gentamicin, tobramycin. Isolates were investigated by multilocus sequence typing (MLST), macrorestriction analysis of genomic DNA (PFGE) and class 1 integron typing. Nine MDR isolates belonged to multilocus seuence type (ST) 235, eight to ST 175 and each of the remaining isolates yielded a unique ST. The STs differed from each other in at least three alleles. Isolates of the same ST had highly similar PFGE profiles. Integrons were found in all MDR isolates but in none of the susceptible controls. Seven isolates with ST 235 harbored the same integron with a 1.9 kb variable region, while seven isolates with ST 175 shared an integron with a 1.6 kb variable region. Each of the remaining isolates yielded a unique integron. The high prevalence of antibiotic resistance in Czech isolates of P. aeruginosa is likely to be associated with the spread of MDR clones, one of which (ST 235) belongs to international clonal complex CC11.

We previously showed that the opportunistic nosocomial pathogen Acinetobacter baumannii is able to sense and respond to light via BlsA, a BLUF (Blue-Light-sensing Using FAD)-domain photoreceptor protein. Here, we extend our previous studies showing that light regulation is not restricted to A. baumannii, but rather widespread within the genus Acinetobacter. First, we found that blue light modulates motility and biofilm formation in many species of the genus, including members of the Acinetobacter calcoaceticus-A. baumannii complex. In many of these species blue light acts as a key factor guiding the decision between motility or sessility at 24°C, whereas in A. baumannii, light inhibits both motility and biofilm formation. We also show that light regulation of motility occurred not only at 24°C but also at 37°C in non-A. baumannii species, contrasting the situation of A. baumannii which only shows photoregulation at 24°C. Second, we show that Acinetobacter baylyi (strain ADP1) BLUF-photoreceptors can functionally replace in vivo the A. baumannii 17978 BlsA protein and that the pathways leading to biofilm formation are inversely regulated at 24°C between these two microorganisms. Finally, we found the presence of predicted genes coding BLUF-containing proteins in all Acinetobacter sequenced genomes, even though the copy number is variable among them. Phylogenetic analysis suggests a common origin for all BLUF domains present in members of this genus, and could distinguish well-differentiated clusters that group together BLUF homologs from different species, a situation particularly clear for members of the ACB complex. Despite a role played by these BLUF domain-containing proteins in the photoregulation observed in the members of the genus Acinetobacter is a likely scenario given our findings in A. baumannii and A. baylyi, further research will contribute to confirm this possibility.

To assess the species of the genus Aeromonas in 178 strains isolated from human and animal materials and from the environment (water and hospital environment), examined along with 11 type strains of the best known species, a key was used the basis of which are in addition to biochemical tests defining the genus Aeromonas 12 tests: Voges-Proskauer, lysine decarboxylase, gas from glucose, haemolysis, gluconate oxidation, elastase, arabinose, mannose, saccharose, salicine, esculine hydrolysis, arbutine. Three tests--salicine, esculine hydrolysis, arbutine--differentiate A. hydrophila (2-3 positive) from A. sobria (0-1 positive).

... oxygen as the final electron acceptor in the respiratory pathway, fail to grow anaerobically at all ... 37°C, although some strains, among them many of the pink-pigmented species, grow better at ... Using API 20NE, 54% of the isolates were assigned to species level, 7% were assigned ...

The aims of the study were to characterize isolates of Bartonella henselae and to determine the prevalence of bacteremic domestic cats in urban and suburban parts of Prague, Czech Republic. Five (18%) gram-negative fastidious bacterial single-cat isolates were recovered from 27 hemocultures incubated without previous freezing. Four of these isolates originated from flea infested stray cats (n=6) and one from a shelter cat without any ectoparasites (n=21). None of the 34 previously frozen specimens from flea free pet cats yielded any bacteria. All five isolates were catalase and oxidase negative. Their enzymatic activity, RFLP profile of citrate synthetase gene (gltA) and DNA-DNA hybridization results were typical of B. henselae. According to their PvuII and BglI ribotypes the isolates could be allocated to two homogeneous groups. Ribotype HindIII and RFLP of 16S-23S rRNA spacer region analysis gave unique profiles different from those of Bartonella quintana, Bartonella elizabethae and Bartonella clarridgeiae. The 16S rRNA type-specific amplification revealed an identical profile typical of B. henselae genotype II for all the cat isolates studied. Pulsed-field gel electrophoresis (PFGE) assigned a different profile to each of the isolates studied. Determination of the enzymatic activity, RFLP of gltA gene, RFLP of 16S-23S rRNA spacer region, and HindIII ribotype could be efficient tools for identification of B. henselae isolates. Ribotyping (PvuII, BglI), 16S rRNA typing and PFGE may be useful methods to prospect ecology and epidemiology of the agent.

To investigate the nature of aminoglycoside resistance of clinically important multidrug-resistant (MDR) Acinetobacter baumannii strains from the Czech Republic. Seventy MDR hospital strains from 1991-2001 classified into pan-European clones I (n = 41) and II (n = 21) and a group of other MDR strains (n = 8) were studied. Fifteen other strains wild-type susceptible to aminoglycosides were used as controls. Susceptibility to aminoglycosides was determined by the disk diffusion and agar dilution tests. The presence of six genes encoding different aminoglycoside-modifying enzymes (AME) and of the adeB gene encoding a component of the AdeABC efflux pump was detected using PCR. MDR strains showed the following pattern of resistance to aminoglycosides: kanamycin (93 %; inhibition zone diameters < or =13 mm), gentamicin (87 %; MICs > or =16 mg/l), amikacin (47 %; MICs > or =64 mg/l), tobramycin (31 %; MICs > or = 16 mg/l) and netilmicin (17 %; MICs ? 32 mg/l). Forty-six (66 %) MDR strains had netilmicin MICs of 8-16 mg/l in contrast to 0.5-1.0 mg/l found for control susceptible strains. The presence of at least one of the following AME genes was detected in 66 (94 %) MDR strains: aphA1 (n = 56), aacC1 (n = 52), aphA6 (n = 39) and aadB (n = 20). The presence of these genes and phenotypes of resistance to kanamycin, gentamicin, tobramycin and amikacin were in good agreement. The genes encoding netilmicin-modifying enzymes (aacC2, aacA4) were not detected in any strain. Fifty-six (80 %) MDR strains comprised two to four different AME genes in eight combinations. Clones I and II shared all of the genes studied (with the exception of aadB not detected in clone I). The gene adeB was found in all MDR strains and eight of 15 control susceptible strains. Clinically relevant aminoglycoside resistance of Czech A. baumannii strains is significantly associated with the genes encoding enzymatic modification of kanamycin, gentamicin, amikacin and tobramycin. These genes can spread horizontally and emerge in different combinations leading to high-level resistance to multiple aminoglycosides. The AdeABC pump is likely to play a role in the intermediate susceptibility to netilmicin but further study is needed in this regard.

In their recent paper, Kilic et al. (2) reported on a hospital outbreak of bloodstream infections caused by a non-glucose-acidifying Acinetobacter strain. The authors showed that this strain (AK001) was phenotypically most similar to Acinetobacter ursingii but had two properties ...