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PubMed2001

[Community-acquired Acinetobacter baumannii pneumonia].

We report two cases of community-acquired Acinetobacter baumannii pneumonia. Although most infections occur in hospitalized patients, a few cases of community-acquired pneumonia have been described. This disease occurs predominantly in men, and is often associated with underlying conditions such as cigarette smoking, alcohol abuse, diabetes mellitus, and chronic pulmonary diseases. Community-acquired Acinetobacter baumannii pneumonia cases are generally reported from tropical areas, especially during wet season. Microbiological identification in blood or sputum can be difficult because of frequent misinterpretation and possible confusion with Staphylococcus or Haemophilus infuenzae or neisseriae. Early antibiotherapy is required because of the fulminant clinical course, with approximatively 50% fatality rate.

Acinetobacter Infections↗
PubMed1997

PCR-amplified 16S and 23S rDNA restriction analysis for the identification of Acinetobacter strains at the DNA group level.

The genus Acinetobacter is phenotypically rather homogeneous, but genotypically heterogeneous. In this study, a simple method based on restriction analysis of a PCR-amplified large fragment (4.5 kb) of most of the ribosomal operon (16S and 23S ribosomal genes and the spacer in-between) was investigated. Sixty-seven collection strains belonging to the 20 DNA groups proposed until 1993 were studied. Using the enzyme Sau3AI, 25 DNA profiles were obtained. Strains belonging to DNA groups 1, 3, 6, TU13 and TU15 showed two profiles each, and DNA groups 4, 5 and 7 showed profiles with variants showing less intensive additional bands. The remaining 12 groups showed 12 different profiles. The profiles obtained were DNA-group-specific except for one profile which was shared between the unnamed DNA group 3 and a rarely encountered genotypically related DNA group. These two DNA groups could be separated by using the enzyme Hinf1. Twenty-five additional clinical isolates previously characterized by standard DNA-DNA hybridization were selected in a double-blind fashion for identification at the DNA group level to check the reliability of the assay. All strains were correctly identified at the DNA group level. PCR-amplified 16S and 23S rDNA restriction analysis is both an accurate and rapid method for the identification of Acinetobacter at the DNA group level.

Acinetobacter↗
PubMed2001

Detection and characterization of quorum sensing signal molecules in Acinetobacter strains.

Quorum sensing is a widespread regulatory mechanism among Gram-negative bacteria. In this study, Acinetobacter strains were assayed for the presence of quorum sensing signal molecules capable of activating N-acylhomoserine lactone biosensors. By using an Agrobacterium tumefaciens reporter strain it was shown that all the cultures produced two to four detectable signal molecules with different chromatographic patterns. In A. calcoaceticus BD413 supernatants four compounds were detected in a time-dependent manner, and maximal activity was reached at stationary phase. The number of signal molecules was dependent on medium composition; typically, cultures in minimal medium displayed one or two more signals, as compared to complex medium. None of the Acinetobacter supematants showed autoinduction activity with an Chromobacterium violaceum reporter strain, neither in direct or competition assays.

Acinetobacter↗
PubMed1998

Eradication of Acinetobacter baumannii by photosensitized agents in vitro.

The photodynamic effects of photosensitizers on Acinetobacter baumannii were studied. These Gram negative bacteria have recently been implicated in various infections, mainly acquired in hospitals. They have outstanding characteristics of multidrug high resistance to antimicrobial agents. The best photodynamic effect was obtained when A. baumannii cultures were treated with light activated deuteroporphyrin (Dp) at a concentration of 34 mumoles l-off and polymyxin nonapeptide (PMNP) at a concentration of 200 mumoles l-1. At these concentrations the culture in brain heart infusion (BHI) broth was found to be sterile after l h of treatment. Some inhibition was also obtained under the same conditions with Cd-texaphyrin (Cd-Tx) in the presence of PMNP. Treatment with various other photosensitizers in the presence of PMNP exhibited only marginal antibacterial activity. The cationic photosensitizer tetra-methylpyridyl porphine (TMPyP) did not exhibit any photodynamic effect on A. baumannii when illuminated during its growth in BHI broth. Bacteria grown in nutrient broth or suspended in saline and treated by TMPyP resulted in a significant photoinactivation by the sensitizer alone even in the absence of PMNP. It was found that a high concentration of the proteins present in BHI or in serum prevent TMPyP from acting as a photosensitizer against A. baumannii. Bovine serum albumin at the same high protein concentration prevents Dp (in the presence of PMNP) to act as a photosensitizer. The anionic photosensitizer tetra-sulfonatophenyl porphine (TPPS4) did not show any photodynamic effect in high or low protein media. In this study it was found that despite the high resistance of the Acinetobacter baumannii to antibiotics, these bacteria can be significantly photoinactivated by treatment with either Dp + PMNP or TMPyP in low protein content environments. When the protein concentration is high photoinactivation efficiency depends on the type of protein present in the medium.

Acinetobacter↗
PubMed2000

Purification and catalytic properties of two catechol 1,2-dioxygenase isozymes from benzoate-grown cells of Acinetobacter radioresistens.

Two catechol 1,2-dioxygenase (C1,2O) isozymes (IsoA and IsoB) have been purified to homogeneity from a strain of Acinetobacter radioresistens grown on benzoate as the sole carbon and energy source. IsoA and IsoB are both homodimers composed of a single type of subunit with molecular mass of 38,600 and 37,700, Da respectively. In conditions of low ionic strength, IsoA can aggregate as a trimer, in contrast to IsoB, which maintains the dimeric structure, as also supported by the kinetic parameters (Hill numbers). IsoA is identical to the enzyme previously purified from the same bacterium grown on phenol, whereas the IsoB is selectively expressed using benzoate as carbon source. This is the first evidence of the presence of differently expressed C1,2O isozymes in A. radioresistens or more generally of multiple C1,2O isozymes in benzoate-grown Acinetobacter cells. Purified IsoA and IsoB contain approximately 1 iron(III) ion per subunit and both show electronic absorbance and EPR features typical of Fe(III) intradiol dioxygenases. The kinetic properties of the two enzymes such as the specificities toward substituted catechols, the main catalytic parameters, and their behavior in the presence of different kind of inhibitors are, unexpectedly, very similar, in contrast to most of the previously known dioxygenase isozymes.

Acinetobacter↗
PubMed2003

Long-term analysis of diesel fuel consumption in a co-culture of Acinetobacter venetianus, Pseudomonas putida and Alcaligenes faecalis.

The dynamics of a microbial population isolated from superficial waters of Venice Lagoon and the ability to utilise diesel fuel (n-alkanes mixture C12-C28) as the sole carbon and energy source were studied in a long-term reconstruction experiment. The reconstructed microbial population consisted of three bacterial strains belonging to the species Acinetobacter venetianus, Pseudomonas putida, and Alcaligenes faecalis, which were able to oxidise n-alkanes to alkanoates, n-alkanols to alkanoates, or only n-alkanoates, respectively. Three different approaches: plate counting, cell counting by epifluorescence microscopy with DAPI staining, and by fluorescence in situ hybridisation (FISH) by using a probe conjugate with fluoresceine isothiocyanate specifically targeted towards the 16S rRNA of bacteria belonging to the genus Acinetobacter were used to monitor the growth of the bacterial population. The growth of A. venetianus was stimulated by the presence of other strains, suggesting a beneficial interaction. After the first week of growth A. venetianus cells formed aggregates, as confirmed by confocal microscopy (CLSM), which allowed them to be distinguished from free cells. A relationship between cell number and measured areas (microm2) per aggregate was found. Each cell presented an average surface of 1.21 microm2. Each aggregate was formed by a cellular monolayer biofilm consisting of up to several thousands of cells. The A. venetianus aggregates increased in number and size over time, but after two weeks fragmentation events, which had a beneficial effect on the growth of P. putida and A. faecalis, occurred.

Acinetobacter↗
PubMed1997

Diversity of isolates of Acinetobacter from activated sludge systems based on their whole cell protein patterns.

Whole cell protein extracts from strains of the currently recognized genomic species of Acinetobacter, together with those from a range of isolates of several genomic species identified using the Biolog system and obtained from a biological nutrient-removal activated sludge plant were analysed by SDS-PAGE. The dendrograms obtained after numerical analysis for the known genomic species generally supported the taxonomic relationships suggested from earlier DNA-DNA hybridisation data. In some cases the activated sludge isolates identified to genomic species level clustered closely with the corresponding genomic species reference strains, although isolates 5 and 8/9 were scattered throughout the dendrogram. Considerable variations were seen in the protein patterns of the 27 different environmental isolates of genomic species 7 that were analysed. Three unidentified Acinetobacter isolates examined formed their own subcluster.

Acinetobacter↗
PubMed2003

Genome plasticity in Acinetobacter: new degradative capabilities acquired by the spontaneous amplification of large chromosomal segments.

In Acinetobacter sp. ADP1, growth on benzoate requires regulation of the cat genes by two transcriptional activators. Here, mutants were obtained from a strain lacking both activators by selecting for growth on benzoate medium. The mutants, which arose within 3 weeks at a frequency of approximately 10-8, carried amplified chromosomal regions (amplicons) encompassing the cat genes. Multiple occurrences of low-level expression of catA and the catBCIJFD operon provided sufficient transcription for growth. The amplicons of four independently isolated mutants varied in size from approximately 30-100 kbp of the normally 3.8 Mbp chromosome. Mutants had approximately 10-20 copies of an amplicon in adjacent head-to-tail orientations. At the amplicon's chromosomal endpoint, an atypical junction juxtaposed normally distant DNA regions from opposite sides of the cat genes. The sequences of these junctions revealed the precise recombination sites underlying amplification. Additionally, amplicon stability was evaluated in the absence of selective pressure. The natural competence of Acinetobacter for transformation by linear DNA has allowed the development of a powerful new model system for investigating chromosomal rearrangements and for engineering DNA amplifications for wide-ranging applications. The frequent spontaneous amplification of these large chromosomal segments demonstrated the importance of supra-operonic gene clustering in the evolution of catabolic pathways.

Acinetobacter↗
PubMed1999

Phenol hydroxylase from Acinetobacter radioresistens is a multicomponent enzyme. Purification and characterization of the reductase moiety.

This paper reports the isolation and characterization of phenol hydroxylase (PH) from a strain belonging to the Acinetobacter genus. An Acinetobacter radioresistens culture, grown on phenol as the only carbon and energy source, produced a multicomponent enzyme system, located in the cytoplasm and inducible by the substrate, that is responsible for phenol conversion into catechol. Because of the wide diffusion of phenol as a contaminant, the present work represents an initial step towards the biotechnological treatment of waste waters containing phenol. The reductase component of this PH system has been purified and isolated in large amounts as a single electrophoretic band. The protein contains a flavin cofactor (FAD) and an iron-sulfur cluster of the type [2Fe-2S]. The function of this reductase is to transfer reducing equivalents from NAD(P)H to the oxygenase component. In vitro, the electron acceptors can be cytochrome c as well as other molecules such as 2, 6-dichlorophenolindophenol, potassium ferricyanide, and Nitro Blue tetrazolium. The molecular mass of the reductase was determined to be 41 kDa by SDS/PAGE and 38.8 kDa by gel permeation; its isoelectric point is 5.8. The N-terminal sequence is similar to those of the reductases from A. calcoaceticus NCIB 8250 (10/12 identity) and Pseudomonas CF600 (8/12 identity) PHs, but much less similar (2/12 identity) to that of benzoate dioxygenase reductase from A. calcoaceticus BD413. Similarly, the internal peptide sequence of the A. radioresistens PH reductase displays a good level of identity (9/10) with both A. calcoaceticus NCIB 8250 and Pseudomonas CF600 PH reductase internal peptide sequences but a poorer similarity (3/10) to the internal peptide sequence of benzoate dioxygenase reductase from A. calcoaceticus BD413.

Acinetobacter↗
PubMed2001

Pyrolysis mass spectrometry (PyMS) and 16S-23S rDNA spacer region fingerprinting suggests the presence of novel acinetobacters in activated sludge.

Screening of large numbers of Acinetobacter spp. from activated sludge systems with Pyrolysis Mass Spectrometry (PyMS) showed that many did not cluster tightly with the currently described genomic species which have been obtained mainly from clinical sources. Selected isolates were then genotypically fingerprinted using their 16S-23S rDNA spacer region, and again the data revealed considerable differences in the genomic fingerprints of many of these activated sludge isolates to the predominantly clinical genomic species. In fact, few could be identified from them. The possibility that the current speciation within this genus is not adequate to encompass all these environmental isolates is addressed in relation to the methods used to study the population dynamics of Acinetobacter in activated sludge.

Acinetobacter↗
PubMed2001

DNA polynucleotide probes generated from representatives of the genus Acinetobacter and their application in fluorescence in situ hybridization of environmental samples.

The application of rRNA directed polynucleotide probes carrying multiple labels facilitates the detection of target cells by fluorescence in situ hybridizations and allows specific enrichment by cell fishing. So far, exclusively RNA transcript probes have been used. To reduce the effort in the preparation of the polynucleotides and to enhance their stability, DNA probes matching a part of the highly variable domain III on the 23S rRNA were constructed by amplification of the target region using PCR. Fluorescent labeling was achieved by incorporation of Cy3-labeled desoxyribonucleotides in the amplification. DNA polynucleotide probes were constructed for the seven validly described Acinetobacter species. Amplified domain III rDNA of A. baumannii and A. calcoaceticus could be readily applied as species specific probe. In addition, rDNA fragments could be used to recognize two groups of species, one comprising A. haemolyticus, A. junii and A. radioresistens and the other one A. lwoffii and A. johnsonii. Acinetobacter baumannii cells, some of them occurring in filaments, could be detected by in situ hybridization in native samples of activated sludge.

Acinetobacter↗
PubMed1999

Distribution of Acinetobacter baumannii in a neurointensive care unit.

In a 1-month prospective case-matched study we found Acinetobacter baumannii was a prevailing microbe simultaneously colonizing respiratory tract and skin of neurointensive care unit patients who stayed in our neurointensive care unit for more than 3 d. A. baumannii was not isolated from healthy case-matched controls. Based on their phenotypic properties and the results of amplified ribosomal DNA restriction analysis the 12 strains of Acinetobacter spp. isolated were identified as belonging to DNA group 2 (A. baumannii). For epidemiological typing, Biolog system results of 95-carbon source oxidation, antibiograms and restriction endonuclease analysis were used. One predominant A. baumannii strain was found in all colonized patients, skin and respiratory tract were found mainly to be colonized with the same strain. The starting point of A. baumannii colonization seemed to vary with the individual patient. Environmental strains were different from patients' strains: they were metabolically more active, more resistant and had a different restriction endonuclease analysis profile.

Acinetobacter↗
PubMed1989

The inanimate environment of an intensive care unit as a potential source of nosocomial bacteria: evidence for long survival of Acinetobacter calcoaceticus.

Environmental surface and personnel hand impression cultures were obtained during 13 sampling periods in the University of Virginia Pediatric Intensive Care Unit to document potential reservoirs of nosocomial pathogens. In 78 environmental cultures Staphylococcus aureus was found eight times and gram-negative bacilli ten times. The patient chart cover was the most commonly contaminated surface. Acinetobacter calcoaceticus was found in five of ten cultures positive for gram-negative bacilli. Thirty of 59 hand cultures were positive for S aureus and gram-negative bacilli; nurses and residents had both, respiratory therapists only gram-negative bacilli, and A calcoaceticus was the most commonly isolated bacterium of potentially nosocomial significance (14/30). Laboratory investigation of bacterial survival revealed that gram-negative bacilli survived on a dry formica surface from a few hours up to three days but Acinetobacter survived up to 13 days. Since A calcoaceticus has been implicated in many nosocomial infections, its long survival on a dry surface may be an additional factor in its transmission in hospitals and suggests that more attention be paid to environmental surfaces as a source of significant nosocomial pathogens.

Acinetobacter↗
PubMed1986

Comparative activity of imipenem, ceftazidime and cefotaxime against Acinetobacter calcoaceticus.

Imipenem was the most active drug against Acinetobacter, even against strains possessing beta-lactamases. MICs of imipenem for 50% and 90% of the A. calcoaceticus (var. anitratum) were 0.28 and 0.61 mg/l respectively whereas for ceftazidime they were 4.4 and 10.0, and for cefotaxime, 7.9 and 22.2 mg/l. No change occurred in the in-vitro activity of imipenem against Acinetobacter, during the period 1980-1985. Geometric mean MICs in 1985 were 0.33 mg/l for A. calcoaceticus var. anitratum; the values were lower for A. calcoaceticus var. lwoffi. Ceftazidime and cefotaxime MICs were also stable from 1981 to 1985, but the values were higher, geometric mean MICs being 5.3 and 10.1 mg/l respectively. MBCs of imipenem for A. calcoaceticus var. anitratum ranged from 0.194 to 0.35 mg/l; the ratio MBC/MIC was 1.17.

Acinetobacter↗
PubMed1988

Distribution of beta-lactamases and phenotype analysis in clinical strains of Acinetobacter calcoaceticus.

One hundred clinical strains of Acinetobacter calcoaceticus isolated from 1981 to 1986 were screened for enzymatic resistance to beta-lactam antibiotics. Fourteen beta-lactam antibiotics were tested and four phenotypes were defined on the basis of enzymatic resistance and of susceptibility to the following beta-lactams: ticarcillin, piperacillin, cefotaxime, and ceftazidime. The resistance of A. calcoaceticus to beta-lactam antibiotics was predominantly due to beta-lactamases, which were produced by 81% of the strains. In most (71%) of the beta-lactamase producing strains, a penicillinase of the TEM type was observed; in 9% of the strains, all isolated since 1985, a CARB-type penicillinase with a pI 6.3 was observed. The presence of a cephalosporinase type enzyme was detected in acinetobacter strains isolated since 1981 and its incidence increased in 1986. Multiple beta-lactamases (penicillinase plus cephalosporinase) were observed in 32% of the strains.

Acinetobacter↗
PubMed2005

Characterization of a 2.6 kbp variable region within a class 1 integron found in an Acinetobacter baumannii strain isolated from a horse.

OBJECTIVES: A complete gene cassette contained in a class 1 integron from a multidrug-resistant (MDR) isolate of Acinetobacter baumannii cultured from a horse was characterized by molecular methods. METHODS: Template genomic DNA purified from the A. baumannii isolate was investigated by PCR. A gene cassette-associated amplicon was detected and completely characterized. RESULTS: A 2.6 kbp DNA fragment containing four gene cassettes was amplified from the MDR A. baumannii isolate. Sequence analysis showed it was similar to sequences recently reported in Klebsiella pneumoniae, Serratia marcescens and an Escherichia coli plasmid p1658/97 which conferred aminoglycoside resistance. Aminoglycoside resistance-encoding genes aacC1 and aadA1 were located within the 2.6 kbp amplicon, separated by two open reading frames (ORFs) coding for unknown products. This cassette structure (and some variants) was identified in unrelated Acinetobacter spp. from human sources, based on sequence comparisons of the current databases. CONCLUSIONS: Identification of a complete class 1 integron in an equine isolate of A. baumannii suggests that the screening of isolates from animals for these elements should be considered, as this information could influence the selection of chemotherapeutic agents.

Acinetobacter baumannii↗
PubMed2006

Multidrug efflux inhibition in Acinetobacter baumannii: comparison between 1-(1-naphthylmethyl)-piperazine and phenyl-arginine-beta-naphthylamide.

OBJECTIVES: 1-(1-Naphthylmethyl)-piperazine (NMP) has been shown to reverse multidrug resistance (MDR) in Escherichia coli overexpressing RND-type efflux pumps but there are no data on its activity in non-fermenters like Acinetobacter. METHODS: Antimicrobial susceptibility in the absence and presence of NMP and, for comparison, phenyl-arginine-beta-naphthylamide (PAbetaN), another putative efflux pump inhibitor (EPI), was tested in laboratory and mutant strains with differing intracellular dye accumulation and expression of adeB, and in clinical isolates of Acinetobacter baumannii. RESULTS: Based on a 4-fold or greater MIC reduction, the effects of both EPIs at low concentrations (25 mg/L) were limited. PAbetaN had a highly selective action on the reduction in the MIC of rifampicin and clarithromycin. At a higher concentration of the putative EPIs (100 mg/L), NMP was more active than PAbetaN. This effect was not limited to strains with adeB overexpression, but affected the susceptibility to linezolid, chloramphenicol and tetracycline most, and was enhanced in clinical isolates with reduced fluoroquinolone susceptibility. CONCLUSION: NMP can partially reverse MDR in A. baumannii and differs substantially in its activity from that of PAbetaN.

Acinetobacter baumannii↗
PubMed1999

Random mutagenesis by recombinational capture of PCR products in Bacillus subtilis and Acinetobacter calcoaceticus.

We describe a general method for random mutagenesis of cloned genes by error-prone PCR or DNA shuffling that eliminates the need for post-amplification subcloning following each cycle of mutagenesis. This method exploits the highly efficient and recombinogenic nature of DNA uptake during natural transformation in the Gram-positive bacterium Bacillus subtilis and the Gram-negative bacterium Acinetobacter calcoaceticus. Plasmid systems were designed that allow capture of PCR-amplified DNA fragments by marker-replacement recombination with a structurally similar helper plasmid resident in the transformation recipient. This recombination event simultaneously transfers the amplified sequences into the helper plasmid and restores the integrity of a drug resistance gene, thereby affording a direct selection for fragment capture. Although this strategy was sufficiently effective to permit recovery in B. subtilis of up to 10(3) transformants/microgram of PCR product, equivalent plasmid systems were approximately 100 times more efficient in A.calcoaceticus. Acinetobacter calcoaceticus also offers the advantage of essentially constitutive transformation competence in ordinary complex broth, such as LB, in contrast to two-step growth in semi-synthetic media required for optimal transformation of B.subtilis.

Acinetobacter calcoaceticus↗