Species Distribution of Clinical Acinetobacter Isolates Revealed by Different Identification Techniques

A total of 2582 non-duplicate clinical Acinetobacter spp. isolates were collected to evaluate the performance of four identification methods because it is important to identify Acinetobacter spp. accurately and survey the species distribution to determine the appropriate antimicrobial treatment. Phenotyping (VITEK 2 and VITEK MS) and genotyping (16S rRNA and rpoB gene sequencing) methods were applied for species identification, and antimicrobial susceptibility test of imipenem and meropenem was performed with a disk diffusion assay. Generally, the phenotypic identification results were quite different from the genotyping results, and their discrimination ability was unsatisfactory, whereas 16S rRNA and rpoB gene sequencing showed consistent typing results, with different resolution. Additionally, A. pittii, A. calcoaceticus and A. nosocomialis, which were phylogenetically close to A. baumannii, accounted for 85.5% of the non-A. baumannii isolates. One group, which could not be clustered with any reference strains, consisted of 11 isolates and constituted a novel Acinetobacter species that was entitled genomic species 33YU. None of the non-A. baumannii isolates harbored a bla _OXA-51-like gene, and this gene was disrupted by ISAba19 in only one isolate; it continues to be appropriate as a genetic marker for A. baumannii identification. The resistance rate of non-A. baumannii isolates to imipenem and/or meropenem was only 2.6%, which was significantly lower than that of A. baumannii. Overall, rpoB gene sequencing was the most accurate identification method for Acinetobacter species. Except for A. baumannii, the most frequently isolated species from the nosocomial setting were A. pittii, A. calcoaceticus and A. nosocomialis.     

Combination of ARDRA and RAPD genotyping techniques in identification of Acinetobacter spp. genomic species

A total of 10 non-repetitive multi-drug-resistant Acinetobacter strains were collected. With reference to A. calcoaceticus (ATCC23055), A. baumannii (ATCC19606), A. lwoffii (ATCC17986), and A. junii (NCTC5866),DNA fingerprint technique, amplified ribosomal DNA restriction analysis (ARDRA), and random amplified polymorphism DNA (RAPD) were carried out to identify the genomic species of Acinetobacter spp. The distances between them were calculated by the unweighted pair group method with arithmetic (UPGMA). Genotypes of Acinetobacter spp. were effectively classified and an A. junii together with nine A. baumannii isolates was genomically identified. The combination of ARDRA and RAPD DNA-fingerprint technique shows high complementarity, and could be a useful tool in Acinetobacter genomic species identification. __________ Translated from Microbiology, 2007, 34(2): 303–306 [译自:微生物学通报]     

The Clinical Characteristics,Carbapenem Resistance,and Outcome of Acinetobacter Bacteremia According to Genospecies

Background

Few clinical data are available on the relationship between genospecies and outcome of Acinetobacter bacteremia, and the results are inconsistent. We performed this study to evaluate the relationship between genospecies and the outcome of Acinetobacter bacteremia.

Methods

Clinical data from 180 patients who had Acinetobacter bacteremia from 2003 to 2010 were reviewed retrospectively. The genospecies were identified by rpoB gene sequence analysis. The clinical features and outcomes of 90 patients with A. baumannii bacteremia were compared to those of 90 patients with non-baumannii Acinetobacter bacteremia (60 with A. nosocomialis, 17 with Acinetobacter species “close to 13 TU”, 11 with A. pittii, and two with A. calcoaceticus).

Results

A. baumannii bacteremia was associated with intensive care unit-onset, mechanical ventilation, pneumonia, carbapenem resistance, and higher APACHE II scores, compared to non-baumannii Acinetobacter bacteremia (P<0.05). In univariate analyses, age, pneumonia, multidrug resistance, carbapenem resistance, inappropriate empirical antibiotics, higher APACHE II scores, and A. baumannii genospecies were risk factors for mortality (P<0.05). Multivariate analysis revealed A. baumannii genospecies (OR, 3.60; 95% CI, 1.56–8.33), age, pneumonia, and higher APACHE II scores to be independent risk factors for mortality (P<0.05).

Conclusion

A. baumannii genospecies was an independent risk factor for mortality in patients with Acinetobacter bacteremia. Our results emphasize the importance of correct species identification of Acinetobacter blood isolates.     

Interaction of Acinetobacter baumannii 19606 and 1656-2 with Acanthamoeba castellanii

Acinetobacter baumannii is virtually avirulent for healthy people but maintains a high virulence among critically ill patients or immuno-compromised individuals. The ability of A. baumannii to adhere to cells and persist on surfaces as biofilms could be central to its pathogenicity. In the present study, we compared the virulence of the A. baumannii 1656-2 clinical strain, which is able to form a thick biofilm, with the virulence of the A. baumannii type strain (ATCC 19606^T). Acanthamoeba castellanii, a single-celled organism, was used as the host model system to study the virulence of A. baumannii. Compared to A. baumannii ATCC 19606^T, A. baumannii 1656-2 exhibited a higher ability to adhere and invade A. castellanii cells and had a higher killing rate of A. castellanii cells. Furthermore, co-incubation of the amoeba cells and the cell-free supernatant of A. baumannii resulted in the cell death of the amoebae. Heat inactivation or proteinase K treatment of the supernatant did not eliminate its cytotoxicity, suggesting heat stable non-protein factors are responsible for its cytotoxicity to A. castellanii cells. In conclusion, this study for the first time has revealed the capacity of the A. baumannii strain and/or its metabolic products to induce cytotoxicity in A. castellanii cells.     

Insertions or Deletions (Indels) in the rrn 16S-23S rRNA Gene Internal Transcribed Spacer Region (ITS) Compromise the Typing and Identification of Strains within the Acinetobacter calcoaceticus-baumannii (Acb) Complex and Closely Related Members

To determine whether ITS sequences in the rrn operon are suitable for identifying individual Acinetobacter Acb complex members, we analysed length and sequence differences between multiple ITS copies within the genomes of individual strains. Length differences in ITS reported previously between A. nosocomialis BCRC15417^T (615 bp) and other strains (607 bp) can be explained by presence of an insertion (indel 13i/1) in the longer ITS variant. The same Indel 13i/1 was also found in ITS sequences of ten strains of A. calcoaceticus, all 639 bp long, and the 628 bp ITS of Acinetobacter strain BENAB127. Four additional indels (13i/2–13i/5) were detected in Acinetobacter strain c/t13TU 10090 ITS length variants (608, 609, 620, 621 and 630 bp). These ITS variants appear to have resulted from horizontal gene transfer involving other Acinetobacter species or in some cases unrelated bacteria. Although some ITS copies in strain c/t13TU 10090 are of the same length (620 bp) as those in Acinetobacter strains b/n1&3, A. pittii (10 strains), A. calcoaceticus and A. oleivorans (not currently acknowledged as an Acb member), their individual ITS sequences differ. Thus ITS length by itself can not by itself be used to identify Acb complex strains. A shared indel in ITS copies in two separate Acinetobacter species compromises the specificity of ITS targeted probes, as shown with the Aun-3 probe designed to target the ITS in A. pitti. The presence of indel 13i/5 in the ITS of Acinetobacter strain c/t13TU means it too responded positively to this probe. Thus, neither ITS sequencing nor the currently available ITS targeted probes can distinguish reliably between Acb member species.     

Histopathological Analysis of Acinetobacter baumannii Lung Infection in a Mouse Model

Acinetobacter baumannii is the main causative pathogen of nosocomial infections that causes severe infections in the lungs. In this study, we analyzed the histopathological characteristics of lung infection with two strains of A. baumannii (ATCC 19606 and the clinical isolate TK1090) and Pseudomonas aeruginosa PAO-1 in C3H/HeN mice to evaluate the virulence of A. baumannii. Survival was evaluated over 14 days. At 1, 2, 5, or 14 days postinfection, mice of C3H/HeN were sacrificed, and histopathological analysis of lung specimens was also performed. Histopathological changes and accumulation of neutrophils and macrophages in the lungs after infection with A. baumannii and P. aeruginosa were analyzed. Following intratracheal inoculation, the lethality of ATCC 19606- and TK1090-infected mice was lower than that of PAO-1-infected mice. However, when mice were inoculated with a sub-lethal dose of A. baumannii, the lung bacterial burden remained in the mice until 14 days post-infection. Additionally, histopathological analysis revealed that macrophages infiltrated the lung foci of ATCC 19606-, TK1090-, and PAO-1-infected mice. Although neutrophils infiltrated the lung foci of ATCC 19606- and TK1090-infected mice, they poorly infiltrated the lung foci of PAO-1-infected mice. Accumulation of these cells in the lung foci of ATCC 19606- and TK1090-infected mice, but not PAO-1-infected mice, was observed for 14 days post-infection. These results suggest that A. baumannii is not completely eliminated despite the infiltration of immune cells in the lungs and that inflammation lasts for prolonged periods in the lungs. Further studies are required to understand the mechanism of A. baumannii infection, and novel drugs and vaccines should be developed to prevent A. baumannii infection.     

Diversity of Acinetobacter baumannii in Four French Military Hospitals, as Assessed by Multiple Locus Variable Number of Tandem Repeats Analysis

Background

Infections by A. calcoaceticus-A. baumannii (ACB) complex isolates represent a serious threat for wounded and burn patients. Three international multidrug-resistant (MDR) clones (EU clone I-III) are responsible for a large proportion of nosocomial infections with A. baumannii but other emerging strains with high epidemic potential also occur.

Methodology/Principal Findings

We automatized a Multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) protocol and used it to investigate the genetic diversity of 136 ACB isolates from four military hospitals and one childrens hospital. Acinetobacter sp other than baumannii isolates represented 22.6% (31/137) with a majority being A. pittii. The genotyping protocol designed for A.baumannii was also efficient to cluster A. pittii isolates. Fifty-five percent of A. baumannii isolates belonged to the two international clones I and II, and we identified new clones which members were found in the different hospitals. Analysis of two CRISPR-cas systems helped define two clonal complexes and provided phylogenetic information to help trace back their emergence.

Conclusions/Significance

The increasing occurrence of A. baumannii infections in the hospital calls for measures to rapidly characterize the isolates and identify emerging clones. The automatized MLVA protocol can be the instrument for such surveys. In addition, the investigation of CRISPR/cas systems may give important keys to understand the evolution of some highly successful clonal complexes.     

Comparative analysis of fecal microflora of healthy full-term Indian infants born with different methods of delivery (vaginal vs cesarean): <Emphasis Type="Italic">Acinetobacter</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> prevalence in vaginally born infants

In this study fecal microflora of human infants born through vaginal delivery (VB) and through cesarean section (CB) were investigated using culture-independent 16S rDNA cloning and sequencing approach. The results obtained clearly revealed that fecal microbiota of VB infants distinctly differ from those in their counterpart CB infants. The intestinal microbiota of infants delivered by cesarean section appears to be more diverse, in terms of bacteria species, than the microbiota of vaginally delivered infants. The most abundant bacterial species present in VB infants were Acinetobacter sp., Bifidobacterium sp. and Staphylococcus sp. However, CB infant’s fecal microbiota was dominated with Citrobacter sp., Escherichia coli and Clostridium difficile. The intestinal microbiota of cesarean section delivered infants in this study was also characterized by an absence of Bifidobacteria species. An interesting finding of our study was recovery of large number of Acinetobacter sp. consisting of Acinetobacter pittii (former Acinetobacter genomic species 3), Acinetobacter junii and Acinetobacter baumannii in the VB infants clone library. Among these, Acinetobacter baumannii is a known nosocomial pathogen and Acinetobacter pittii (genomic species 3) is recently recognized as clinically important taxa within the Acinetobacter calcoaceticus–Acinetobacter baumannii (ACB) complex. Although none of the infants had shown any sign of clinical symptoms of disease, this observation warrants a closer look.     

Modern Acinetobacter baumannii clinical isolates replicate inside spacious vacuoles and egress from macrophages

Multidrug-resistant Acinetobacter baumannii infections are increasing at alarming rates. Therefore, novel antibiotic-sparing treatments to combat these A. baumannii infections are urgently needed. The development of these interventions would benefit from a better understanding of this bacterium’s pathobiology, which remains poorly understood. A. baumannii is regarded as an extracellular opportunistic pathogen. However, research on Acinetobacter has largely focused on common lab strains, such as ATCC 19606, that have been isolated several decades ago. These strains exhibit reduced virulence when compared to recently isolated clinical strains. In this work, we demonstrate that, unlike ATCC 19606, several modern A. baumannii clinical isolates, including the recent clinical urinary isolate UPAB1, persist and replicate inside macrophages within spacious vacuoles. We show that intracellular replication of UPAB1 is dependent on a functional type I secretion system (T1SS) and pAB5, a large conjugative plasmid that controls the expression of several chromosomally-encoded genes. Finally, we show that UPAB1 escapes from the infected macrophages by a lytic process. To our knowledge, this is the first report of intracellular growth and replication of A. baumannii. We suggest that intracellular replication within macrophages may contribute to evasion of the immune response, dissemination, and antibiotic tolerance of A. baumannii.     

Unusual deprivation of compatible solutes in Acinetobacter baumannii

The opportunistic human pathogen Acinetobacter baumannii is one of the leading causes of nosocomial infections. The high prevalence of multidrug-resistant strains, a high adaptability to changing environments and an overall pronounced stress resistance contribute to persistence and spread of the bacteria in hospitals and thereby promote repeated outbreaks. Altogether, the success of A. baumannii is mainly built on adaptation and stress resistance mechanisms, rather than relying on ‘true’ virulence factors. One of the stress factors that pathogens must cope with is osmolarity, which can differ between the external environment and different body parts of the human host. A. baumannii ATCC 19606^T accumulates the compatible solutes glutamate, mannitol and trehalose in response to high salinities. In this work, it was found that most of the solutes vanish immediately after reaching stationary phase, a very unusual phenomenon. While glutamate can be metabolized, mannitol produced by MtlD is excreted to the medium in high amounts. First results indicate that A. baumannii ATCC 19606^T undergoes a rapid switch to a dormant state (viable but non-culturable) after disappearance of the compatible solutes. Resuscitation from this state could easily be achieved in PBS or fresh medium.     

Immunization with Lipopolysaccharide-Deficient Whole Cells Provides Protective Immunity in an Experimental Mouse Model of Acinetobacter baumannii Infection

The increasing clinical importance of infections caused by multidrug resistant Acinetobacter baumannii warrants the development of novel approaches for prevention and treatment. In this context, vaccination of certain patient populations may contribute to reducing the morbidity and mortality caused by this pathogen. Vaccines against Gram-negative bacteria based on inactivated bacterial cells are highly immunogenic and have been shown to produce protective immunity against a number of bacterial species. However, the high endotoxin levels present in these vaccines due to the presence of lipopolysaccharide complicates their use in human vaccination. In the present study, we used a laboratory-derived strain of A. baumannii that completely lacks lipopolysaccharide due to a mutation in the lpxD gene (IB010), one of the genes involved in the first steps of lipopolysaccharide biosynthesis, for vaccination. We demonstrate that IB010 has greatly reduced endotoxin content (<1.0 endotoxin unit/10⁶ cells) compared to wild type cells. Immunization with formalin inactivated IB010 produced a robust antibody response consisting of both IgG1 and IgG2c subtypes. Mice immunized with IB010 had significantly lower post-infection tissue bacterial loads and significantly lower serum levels of the pro-inflammatory cytokines IL-1β, TNF-α and IL-6 compared to control mice in a mouse model of disseminated A. baumannii infection. Importantly, immunized mice were protected from infection with the ATCC 19606 strain and an A. baumannii clinical isolate. These data suggest that immunization with inactivated A. baumannii whole cells deficient in lipopolysaccharide could serve as the basis for a vaccine for the prevention of infection caused by A. baumannii.     

Acinetobacter baumannii Outer Membrane Vesicles Elicit a Potent Innate Immune Response via Membrane Proteins

Acinetobacter baumannii is increasingly becoming a major nosocomial pathogen. This opportunistic pathogen secretes outer membrane vesicles (OMVs) that interact with host cells. The aim of this study was to investigate the ability of A. baumannii OMVs to elicit a pro-inflammatory response in vitro and the immunopathology in response to A. baumannii OMVs in vivo. OMVs derived from A. baumannii ATCC 19606^T induced expression of pro-inflammatory cytokine genes, interleukin (IL)-1β and IL-6, and chemokine genes, IL-8, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1, in epithelial cells in a dose-dependent manner. Disintegration of OMV membrane with ethylenediaminetetraacetic acid resulted in low expression of pro-inflammatory cytokine genes, as compared with the response to intact OMVs. In addition, proteinase K-treated A. baumannii OMVs did not induce significant increase in expression of pro-inflammatory cytokine genes above the basal level, suggesting that the surface-exposed membrane proteins in intact OMVs are responsible for pro-inflammatory response. Early inflammatory processes, such as vacuolization and detachment of epithelial cells and neutrophilic infiltration, were clearly observed in lungs of mice injected with A. baumannii OMVs. Our data demonstrate that OMVs produced by A. baumannii elicit a potent innate immune response, which may contribute to immunopathology of the infected host.     

<Emphasis Type="Italic">Acinetobacter brisouii</Emphasis> sp. nov., isolated from a wetland in Korea

A bacterial strain 5YN5-8^T was isolated from peat layer on Yongneup in Korea. Cells of strain 5YN5-8^T were strictly aerobic, Gram-negative, coccobacilli, non-spore forming, and non-motile. The isolate exhibited optimal growth at 28°C, pH 7.0, and 0–1% NaCl. Results of 16S rRNA gene sequence analyses indicated a close relationship of this isolate to Acinetobacter calcoaceticus (97.8% similarity for strain DSM 30006^T). It also exhibited 94.4–97.8% 16S rRNA gene sequence similarities to the validly published Acinetobacter species. The value for DNA-DNA hybridization between strain 5YN5-8^T and other members of the genus Acinetobacter ranged from 16 to 28%. Predominant cellular fatty acids were C_18:1 ω9c, summed feature 4 containing C_15:0 iso 2-OH and/or C_16:1 ω7c, and C_16:0. The DNA G+C content was 43.9 mol%. Phylogenetic, phenotypic, and chemotaxonomic data accumulated in this study revealed that the isolate could be classified in a novel species of the genus Acinetobacter. The name Acinetobacter brisouii sp. nov. is proposed for the novel species, with 5YN5-8^T (=KACC 11602^T = DSM 18516^T) as the type strain.     

Genomic and Functional Analysis of the Type VI Secretion System in Acinetobacter

The genus Acinetobacter is comprised of a diverse group of species, several of which have raised interest due to potential applications in bioremediation and agricultural purposes. In this work, we show that many species within the genus Acinetobacter possess the genetic requirements to assemble a functional type VI secretion system (T6SS). This secretion system is widespread among Gram negative bacteria, and can be used for toxicity against other bacteria and eukaryotic cells. The most studied species within this genus is A. baumannii, an emerging nosocomial pathogen that has become a significant threat to healthcare systems worldwide. The ability of A. baumannii to develop multidrug resistance has severely reduced treatment options, and strains resistant to most clinically useful antibiotics are frequently being isolated. Despite the widespread dissemination of A. baumannii, little is known about the virulence factors this bacterium utilizes to cause infection. We determined that the T6SS is conserved and syntenic among A. baumannii strains, although expression and secretion of the hallmark protein Hcp varies between strains, and is dependent on TssM, a known structural protein required for T6SS function. Unlike other bacteria, A. baumannii ATCC 17978 does not appear to use its T6SS to kill Escherichia coli or other Acinetobacter species. Deletion of tssM does not affect virulence in several infection models, including mice, and did not alter biofilm formation. These results suggest that the T6SS fulfils an important but as-yet-unidentified role in the various lifestyles of the Acinetobacter spp.     

Acinetobacter strains IH9 and OCI1, two rhizospheric phosphate solubilizing isolates able to promote plant growth,constitute a new genomovar of Acinetobacter calcoaceticus

During a screening of phosphate solubilizing bacteria (PSB) in agricultural soils, two strains, IH9 and OCI1, were isolated from the rhizosphere of grasses in Spain, and they showed a high ability to solubilize phosphate in vitro. Inoculation experiments in chickpea and barley were conducted with both strains and the results demonstrated their ability to promote plant growth. The 16S rRNA gene sequences of these strains were nearly identical to each other and to those of Acinetobacter calcoaceticus DSM 30006^T, as well as the strain CIP 70.29 representing genomospecies 3. Their phenotypic characteristics also coincided with those of strains forming the A. calcoaceticus–baumannii complex. They differed from A. calcoaceticus in the utilization of l-tartrate as a carbon source and from genomospecies 3 in the use of d-asparagine as a carbon source. The 16S–23S intergenic spacer (ITS) sequences of the two isolates showed nearly 98% identities to those of A. calcoaceticus, confirming that they belong to this phylogenetic group. However, the isolates appeared as a separate branch from the A. calcoaceticus sequences, indicating their molecular separation from other A. calcoaceticus strains. The analysis of three housekeeping genes, recA, rpoD and gyrB, confirmed that IH9 and OCI1 form a distinct lineage within A. calcoaceticus. These results were congruent with those from DNA–DNA hybridization, indicating that strains IH9 and OCI1 constitute a new genomovar for which we propose the name A. calcoaceticus genomovar rhizosphaerae.     

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).     

Acinetobacter baumannii Virulence Is Mediated by the Concerted Action of Three Phospholipases D

Acinetobacter baumannii causes a broad range of opportunistic infections in humans. Its success as an emerging pathogen is due to a combination of increasing antibiotic resistance, environmental persistence and adaptation to the human host. To date very little is known about the molecular basis of the latter. Here we demonstrate that A. baumannii can use phosphatidylcholine, an integral part of human cell membranes, as sole carbon and energy source. We report on the identification of three phospholipases belonging to the PLD superfamily. PLD1 and PLD2 appear restricted to the bacteria and display the general features of bacterial phospholipases D. They possess two PLDc_2 PFAM domains each encompassing the HxKx₄Dx₆GS/GGxN (HKD) motif necessary for forming the catalytic core. The third candidate, PLD3, is found in bacteria as well as in eukaryotes and harbours only one PLDc_2 PFAM domain and one conserved HKD motif, which however do not overlap. Employing a markerless mutagenesis system for A. baumannii ATCC 19606^T, we generated a full set of PLD knock-out mutants. Galleria mellonella infection studies as well as invasion experiments using A549 human lung epithelial cells revealed that the three PLDs act in a concerted manner as virulence factors and are playing an important role in host cell invasion.     

Real-Time Fluorescence Loop Mediated Isothermal Amplification for the Detection of Acinetobacter baumannii

Background

Detection of Acinetobacter baumannii has been relying primarily on bacterial culture that often fails to return useful results in time. Although DNA-based assays are more sensitive than bacterial culture in detecting the pathogen, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. In addition, these molecular tools require expensive laboratory instruments. Therefore, establishing molecular tools for field use require simpler molecular platforms. The loop-mediated isothermal amplification method is relatively simple and can be improved for better use in a routine clinical bacteriology laboratory. A simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in the same platform has been developed in recent years. This method is referred to as real-time loop-mediated isothermal amplification. In this study, we attempted to utilize this method for rapid detection of A. baumannii.

Methodology and Significant Findings

Species-specific primers were designed to test the utility of this method. Clinical samples of A. baumannii were used to determine the sensitivity and specificity of this system compared to bacterial culture and a polymerase chain reaction method. All positive samples isolated from sputum were confirmed to be the species of Acinetobacter by 16S rRNA gene sequencing. The RealAmp method was found to be simpler and allowed real-time detection of DNA amplification, and could distinguish A. baumannii from Acinetobacter calcoaceticus and Acinetobacter genomic species 3. DNA was extracted by simple boiling method. Compared to bacterial culture, the sensitivity and specificity of RealAmp in detecting A. baumannii was 98.9% and 75.0%, respectively.

Conclusion

The RealAmp assay only requires a single unit, and the assay positivity can be verified by visual inspection. Therefore, this assay has great potential of field use as a molecular tool for detection of A. baumannii.     

Naturally Transformable Acinetobacter sp. Strain ADP1 Belongs to the Newly Described Species Acinetobacter baylyi

Genotypic and phenotypic analyses were carried out to clarify the taxonomic position of the naturally transformable Acinetobacter sp. strain ADP1. Transfer tDNA-PCR fingerprinting, 16S rRNA gene sequence analysis, and selective restriction fragment amplification (amplified fragment length polymorphism analysis) indicate that strain ADP1 and a second transformable strain, designated 93A2, are members of the newly described species Acinetobacter baylyi. Transformation assays demonstrate that the A. baylyi type strain B2^T and two other originally identified members of the species (C5 and A7) also have the ability to undergo natural transformation at high frequencies, confirming that these five strains belong to a separate species of the genus Acinetobacter, characterized by the high transformability of its strains that have been cultured thus far.     

Isolation and characterization of arsenite-oxidizing bacteria from arsenic-contaminated soils in Thailand

Two hundred and eighty-eight arsenic-resistant bacteria were isolated by an enrichment culture method from a total of 69 arsenic-contaminated soil-samples collected from Dantchaeng district in Suphanburi province (47 samples), and from Ron Phiboon district in Nakhon Sri Thammarat province (22 samples), in Central and Southern Thailand, respectively. Twenty-four of the 288 isolated arsenic-resistant bacteria were found to be arsenite-oxidizing bacteria. On the basis of their morphological, cultural, physiological, biochemical and chemotaxonomic characteristics, and supported by phylogenetic analysis based upon their 16S rRNA gene sequences, they were divided into five groups, within the genera Acinetobacter, Flavobacterium, Pseudomonas, Sinorhizobium and Sphingomonas, respectively. Within genera, phylogenetic analysis using the 16S rRNA gene sequences suggested that they were comprised of at least ten species, five isolates being closely related to known bacteria (Acinetobacter calcoaceticus NCCB 22016^T, Pseudomonas plecoglossicida FPC951^T, Ps. knackmussii B13^T, Sinorhizobium morelense Lc04^T, and Sphingomonas subterranea IFO16086^T). The other five proposed species are likely to be new species closely related to Flavobacterium johnsoniae, Sinorhizobium morelense, Acinetobacter calcoaceticus and Pseudomonas plecoglossicida, but this awaits further characterization for confirmation of the taxonomic status. No overlap in isolated species or strains was observed between the two sites. The strain distribution and characterization are described.