Biosafety assessment of Acinetobacter strains isolated from the Three Gorges Reservoir region in nematode Caenorhabditis elegans.

Acinetobacter has been frequently detected in backwater areas of the Three Gorges Reservoir (TGR) region. We here employed Caenorhabditis elegans to perform biosafety assessment of Acinetobacter strains isolated from backwater area in the TGR region. Among 21 isolates and 5 reference strains of Acinetobacter, exposure to Acinetobacter strains of AC1, AC15, AC18, AC21, A. baumannii ATCC 19606T, A. junii NH88-14, and A. lwoffii DSM 2403T resulted in significant decrease in locomotion behavior and reduction in lifespan of Caenorhabditis elegans. In nematodes, exposure to Acinetobacter strains of AC1, AC15, AC18, AC21, A. baumannii, A. junii and A. lwoffii also resulted in significant reactive oxygen species (ROS) production. Moreover, exposure to Acinetobacter isolates of AC1, AC15, AC18, and AC21 led to significant increase in expressions of both SOD-3::GFP and some antimicrobial genes (lys-1, spp-12, lys-7, dod-6, spp-1, dod-22, lys-8, and/or F55G11.4) in nematodes. The Acinetobacter isolates of AC1, AC15, AC18, and AC21 had different morphological, biochemical, phylogenetical, and virulence gene properties. Our results suggested that exposure risk of some Acinetobacter strains isolated from the TGR region exists for environmental organisms and human health. In addition, C. elegans is useful to assess biosafety of Acinetobacter isolates from the environment.

A point prevalence study to determine the inpatient rate of carbapenemase-producing organisms at a large London NHS Trust.

BACKGROUND: There has been an increase in the number of carbapenemase-producing organisms documented across the UK over the past 10 years. From these, the 'big five' carbapenemases (KPC, OXA-48, IMP, VIM, and NDM) are the most common types reported in the order Enterobacterales, identified from a variety of reactive screening, outbreak, inpatient surveillance, and diagnostic samples. AIM: To perform a point prevalence study to determine the inpatient carriage rate of carbapenemase-producing organisms at Barts Health NHS Trust, which encompasses 2.5 million patients across four London boroughs: Tower Hamlets, Newham, Redbridge, and Waltham Forest. METHODS: Rectal swabs were collected from consenting inpatients, alongside details of the ward's medical specialty, patient's country of birth, history of foreign travel, length of hospitalization, and history of prior hospitalization. Swabs were enriched and subcultured on to mSuperCARBA selective medium. All Enterobacterales, Acinetobacter, and Pseudomonas species were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy and underwent antibiotic susceptibility testing by disc diffusion, according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. All isolates were screened for the 'big five' carbapenemases using a modified version of a published reverse transcriptase-polymerase chain reaction assay. FINDINGS: Of the 977 inpatients tested, 35 CPOs were isolated from 30 patients. NDM was the most frequently detected carbapenemase, followed by OXA-48, with an overall prevalence of 3.1%. Organisms isolated included Klebsiella pneumoniae, Enterobacter cloacae, Proteus mirabilis, and Escherichia coli. Renal and elderly care patients had the highest prevalences of CPOs, whereas the intensive care unit prevalence was low. Statistical analysis found that hospitalization abroad, any previous hospitalization, foreign travel and, specifically, travel to India, Pakistan, and Bangladesh were associated with increased risk of CPO carriage. CONCLUSION: The overall prevalence of CPOs at Barts Health Trust was 3.1%, comprising NDM and OXA-48-type carbapenemases, which is in line with other London-based studies. Renal patients and the elderly had the highest burden of CPOs, whereas previous hospitalization and foreign travel were associated with an increased risk of CPO carriage.

Illumina sequencing of 16S rRNA tag shows disparity in rhizobial and non-rhizobial diversity associated with root nodules of mung bean (Vigna radiata L.) growing in different habitats in Pakistan.

In Rhizobium-legume symbiosis, the nodule is the most frequently studied compartment, where the endophytic/symbiotic microbiota demands critical investigation for development of specific inocula. We identified the bacterial diversity within root nodules of mung bean from different growing areas of Pakistan using Illumina sequencing of 16S rRNA gene. We observed specific OTUs related to specific site where Bradyrhizobium was found to be the dominant genus comprising of 82-94% of total rhizobia in nodules with very minor fraction of sequences from other rhizobia at three sites. In contrast, Ensifer (Sinorhizobium) was single dominant genus comprising 99.9% of total rhizobial sequences at site four. Among non-rhizobial sequences, the genus Acinetobacter was abundant (7-18% of total sequences), particularly in Bradyrhizobium-dominated nodule samples. Rhizobia and non-rhizobial PGPR isolated from nodule samples include Ensifer, Bradyrhizobium, Acinetobacter, Microbacterium and Pseudomonas strains. Co-inoculation of multi-trait PGPR Acinetobacter sp. VrB1 with either of the two rhizobia in field exhibited more positive effect on nodulation and plant growth than single-strain inoculation which favors the use of Acinetobacter as an essential component for development of mung bean inoculum. Furthermore, site-specific dominance of rhizobia and non-rhizobia revealed in this study may contribute towards decision making for development and application of specific inocula in different habitats.

Metagenomic assessment of the interplay between the environment and the genetic diversification of Acinetobacter.

Most bacteria have poorly characterized environmental reservoirs and unknown closely related species. This hampers the study of bacterial evolutionary ecology because both the environment and the genetic background of ancestral lineages are unknown. We combined metagenomics, comparative genomics and phylogenomics to overcome this limitation, to identify novel taxa and to propose environments where they can be isolated. We applied this method to characterize the ecological distribution of known and novel lineages of Acinetobacter spp. We observed two major environmental transitions at deep phylogenetic levels, splitting the genus into three ecologically differentiated clades. One of these has rapidly shifted towards host-association by acquiring genes involved in bacteria-eukaryote interactions. We show that environmental perturbations affect species distribution in predictable ways: bovines have very diverse communities of Acinetobacter, unless they were administered antibiotics, in which case they show highly uniform communities of Acinetobacter spp. that resemble those of humans. Our results uncover the diversity of bacterial lineages, overpassing the limitations of classical cultivation methods and highlight the role of the environment in shaping their evolution.

Costs and benefits of natural transformation in Acinetobacter baylyi.

BACKGROUND: Natural transformation enables acquisition of adaptive traits and drives genome evolution in prokaryotes. Yet, the selective forces responsible for the evolution and maintenance of natural transformation remain elusive since taken-up DNA has also been hypothesized to provide benefits such as nutrients or templates for DNA repair to individual cells. RESULTS: We investigated the immediate effects of DNA uptake and recombination on the naturally competent bacterium Acinetobacter baylyi in both benign and genotoxic conditions. In head-to-head competition experiments between DNA uptake-proficient and -deficient strains, we observed a fitness benefit of DNA uptake independent of UV stress. This benefit was found with both homologous and heterologous DNA and was independent of recombination. Recombination with taken-up DNA reduced survival of transformed cells with increasing levels of UV-stress through interference with nucleotide excision repair, suggesting that DNA strand breaks occur during recombination attempts with taken-up DNA. Consistent with this, we show that absence of RecBCD and RecFOR recombinational DNA repair pathways strongly decrease natural transformation. CONCLUSIONS: Our data show a physiological benefit of DNA uptake unrelated to recombination. In contrast, recombination during transformation is a strand break inducing process that represents a previously unrecognized cost of natural transformation.

Initial Assessment of the Molecular Epidemiology of blaNDM-1 in Colombia.

We report complete genome sequences of four blaNDM-1-harboring Gram-negative multidrug-resistant (MDR) isolates from Colombia. The blaNDM-1 genes were located on 193-kb Inc FIA, 178-kb Inc A/C2, and 47-kb (unknown Inc type) plasmids. Multilocus sequence typing (MLST) revealed that these isolates belong to sequence type 10 (ST10) (Escherichia coli), ST392 (Klebsiella pneumoniae), and ST322 and ST464 (Acinetobacter baumannii and Acinetobacter nosocomialis, respectively). Our analysis identified that the Inc A/C2 plasmid in E. coli contained a novel complex transposon (Tn125 and Tn5393 with three copies of blaNDM-1) and a recombination "hot spot" for the acquisition of new resistance determinants.

Assessment of bacterial acyltransferases for an efficient lipid production in metabolically engineered strains of E. coli.

Microbially produced lipids like triacylglycerols or fatty acid ethyl esters are currently of great interest as fuel replacements or other industrially relevant compounds. They can even be produced by non-oleaginous microbes, like Escherichia coli, upon metabolic engineering. However, there is still much room for improvement regarding the yield for a competitive microbial production of lipids or biofuels. We genetically engineered E. coli by expressing fadD, fadR, pgpB, plsB and 'tesA in combination with atfA from Acinetobacter baylyi. A total fatty acid contents of up to 16% (w/w) was obtained on complex media, corresponding to approximately 9% (w/w) triacylglycerols and representing the highest titers of fatty acids and triacylglycerols obtained in E. coli under comparable cultivation conditions, so far. To evaluate further possibilities for an optimization of lipid production, ten promising bacterial wax ester synthase/acyl-Coenzyme A:diacylglycerol acyltransferases were tested and compared. While highest triacylglycerol storage was achieved with AtfA, the mutated variant AtfA-G355I turned out to be most suitable for fatty acid ethyl ester biosynthesis and enabled an accumulation of approx. 500 mg/L without external ethanol supplementation.

Identification of bacterial protein O-oligosaccharyltransferases and their glycoprotein substrates.

O-glycosylation of proteins in Neisseria meningitidis is catalyzed by PglL, which belongs to a protein family including WaaL O-antigen ligases. We developed two hidden Markov models that identify 31 novel candidate PglL homologs in diverse bacterial species, and describe several conserved sequence and structural features. Most of these genes are adjacent to possible novel target proteins for glycosylation. We show that in the general glycosylation system of N. meningitidis, efficient glycosylation of additional protein substrates requires local structural similarity to the pilin acceptor site. For some Neisserial PglL substrates identified by sensitive analytical approaches, only a small fraction of the total protein pool is modified in the native organism, whereas others are completely glycosylated. Our results show that bacterial protein O-glycosylation is common, and that substrate selection in the general Neisserial system is dominated by recognition of structural homology.

[Fitness costs of blaNDM-1 bearing plasmid pNDM-BJ01 in Acinetobacter lwoffii].

OBJECTIVE: To investigate the fitness costs of the New Delhi Metallo-beta-lactamase-1 (bIaNDM-1) bearing plasmid pNDM-BJ01 in Acinetobacter lwoffii strain 10621, and to evaluate the potentials of the sustainahility and expansions among the Acinetobacter lwoffii population. METHODS: We obtained the spontaneous mutant of A. lwoffii missing pNDM-BJ01 10621NDM-1(-) via serial passages of the wild strain 10621NDM1(-) on the antibiotics-free media. We then compared the in vitro fitness of these two strains by measuring the growth curves, the ability of biofilm formation and the survival rates in nutrient-free PBS buffer. RESULTS: Plasmid pNDM-BJ01 was unstable in A. lwoffii strain 10621, and 11 passages will be enough to get it deleted from the ancestral strain. We found no significant difference in the growth curves either at 26 or 37 degrees C for 10621NDM-1(+) and 10621NDM-1(-). The biofilm formation ability of the plasmid free derivate 10621NDM-1(-) was significant higher than its resistant ancestor 10621NDM-1(+) at 26 degrees C, whereas the latter showed higher ability of biofilm formation at 37 degrees C. Strain 10621NDM-1(+) was vulnerable in the nutrient-free PBS buffer, with less than 5% survival rate on the first day and dying out on the sixth day, whereas 10621NDM-1(-) survived till the seventh day. CONCLUSION: blaNDM-1 bearing plasmid pNDM-BJ01 possesses significant fitness costs in A. lwoffii strain 10621, and it will get depleted easily if the antibiotic press released. pNDM-BJ01-free 10621NDM-1(-) strain has higher fitness than its ancestor, 10621NDM-1(+), which implies the limited expansion potential of plasmid pNDM-BJ01 in A. lwoffii.

Assessment of 16S rRNA gene-based phylogenetic diversity and promising plant growth-promoting traits of Acinetobacter community from the rhizosphere of wheat.

Strains belonging to the genus Acinetobacter and their plant growth-promoting properties have been reported in the literature. However, there is a paucity of information on the diversity of Acinetobacter species associated with the wheat rhizosphere. In the present investigation, Acinetobacter species diversity was assessed in the rhizosphere of wheat from three agricultural fields where different varieties were cultivated. The Acinetobacter species diversity was assessed by DGGE (Denaturing Gradient Gel Electrophoresis) of 16S rRNA gene PCR products amplified from total soil DNA using genus-specific primers. Ac. calcoaceticus, Ac. baumannii, Ac. lwoffii, Ac. baylyi and Acinetobacter sp. were detected in the rhizosphere of wheat. Prevalence of Acinetobacter species in the rhizosphere of wheat was also investigated by a cultivation-dependent approach. Ac. calcoaceticus, Ac. baumannii, Ac. lwoffii and Acinetobacter sp. were isolated on selective media from the same samples. In vitro characterization of Acinetobacter isolates revealed that majority of these bacteria exhibited plant growth-promoting traits such as nitrogen fixation, siderophore production and mineral solubilization. These Acinetobacter strains may play a favorable role in plant growth promotion while residing in the rhizosphere of wheat.

Assessment of transformability of bacteria associated with tomato and potato plants.

Transformation of plant-associated bacteria by plant DNA has never been demonstrated in agricultural fields. In total 552 bacterial isolates from stems of Ralstonia solanacearum-infected and healthy tomato plants and from stems and leaves of healthy potato plants were tested for natural genetic competence using plasmid pSKTG DNA and homologous DNA extracts. Control strain Acinetobacter baylyi ADP1 was transformable with both DNA extracts. No transformable isolates were observed after treatment with plasmid pSKTG DNA. Two isolates, P34, identified as Pseudomonas trivialis and A19, identified as Pseudomonas fragi, were selected on the basis of the consistently higher Rp-resistant CFU numbers after treatment with DNA from Rp-resistant cells than with that from wild-type cells. P34 showed 2.1-fold and A19 1.5-fold higher Rp-resistant CFU numbers after treatment with DNA from homologous Rp-resistant cells versus that from wild-type cells. It is concluded that bacteria capable of in vitro capture and integration of exogenous DNA into their genomes are relatively rare in culturable bacterial communities associated with tomato and potato plants, or that conditions conducive to transformation were not met in transformation assays.

An assessment of the potential of herbivorous insect gut bacteria to develop competence for natural transformation.

Whereas the capability of DNA uptake has been well established for numerous species and strains of bacteria grown in vitro, the broader distribution of natural transformability within bacterial communities remains largely unexplored. Here, we investigate the ability of bacterial isolates from the gut of grass grub larvae (Costelytra zealandica (White); Coleoptera: Scarabaeidae) to develop natural genetic competence in vitro. A total of 37 mostly species-divergent strains isolated from the gut of grass grub larvae were selected for spontaneous rifampicin-resistance. Genomic DNA was subsequently isolated from the resistant strains and exposed to sensitive strains grown individually using established filter transformation protocols. DNA isolated from wild-type strains was used as a control. None of the 37 isolates tested exhibited a frequency of conversion to rifampicin-resistance in the presence of DNA at rates that were significantly higher than the rate of spontaneous mutation to rifampicin-resistance in the presence of wild-type DNA (the limit of detection was approximately < 1 culturable transformant per 10(9) exposed bacteria). To further examine if conditions were conducive to bacterial DNA uptake in the grass grubs gut, we employed the competent bacterium Acinetobacter baylyi strain BD413 as a recipient species for in vivo studies. However, no transformants could be detected above the detection limit of 1 transformant per 10(3) cells, possibly due to low population density and limited growth of A. baylyi cells in grass grub guts. PCR analysis indicated that chromosomal Acinetobacter DNA remains detectable by PCR for up to 3 days after direct inoculation into the alimentary tract of grass grub larvae. Nevertheless, neither transforming activity of the DNA recovered from the alimentary tract of grass grubs larvae nor competence of bacterial cells recovered from inoculated larvae could be shown.

Thio wax ester biosynthesis utilizing the unspecific bifunctional wax ester synthase/acyl coenzyme A:diacylglycerol acyltransferase of Acinetobacter sp. strain ADP1.

The bifunctional wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT) from Acinetobacter sp. strain ADP1 (formerly Acinetobacter calcoaceticus ADP1) mediating the biosyntheses of wax esters and triacylglycerols was used for the in vivo and in vitro biosynthesis of thio wax esters and dithio wax esters. For in vitro biosynthesis, 5'His(6)WS/DGAT comprising an N-terminal His(6) tag was purified from the soluble protein fraction of Escherichia coli Rosetta(DE3)pLysS (pET23a::5'His(6)atf). By employing SP-Sepharose high-pressure and Ni-nitrilotriacetic acid fast-protein liquid chromatographies, a 19-fold enrichment with a final specific activity of 165.2 nmol mg of protein(-1) min(-1) was achieved by using 1-hexadecanol and palmitoyl-CoA as substrates. Incubation of purified 5'His(6)WS/DGAT with 1-hexadecanethiol and palmitoyl-CoA as substrates resulted in the formation of palmitic acid hexadecyl thio ester (10.4% relative specific activity of a 1-hexadecanol control). Utilization of 1,8-octanedithiol and palmitoyl-CoA as substrates led to the formation of 1-S-monopalmitoyloctanedithiol and minor amounts of 1,8-S-dipalmitoyloctanedithiol (59.3% relative specific activity of a 1-hexadecanol control). The latter dithio wax ester was efficiently produced when 1-S-monopalmitoyloctanedithiol and palmitoyl-CoA were used as substrates (13.4% specific activity relative to that of a 1-hexadecanol control). For the in vivo biosynthesis of thio wax esters, the knockout mutant Acinetobacter sp. strain ADP1acr1OmegaKm, which is unable to produce fatty alcohols, was used. Cultivation of Acinetobacter sp. strain ADP1acr1OmegaKm in the presence of gluconate, 1-hexadecanethiol, and oleic acid in nitrogen-limited mineral salts medium resulted in the accumulation of unusual thio wax esters that accounted for around 1.19% (wt/wt) of the cellular dry weight and consisted mainly of oleic acid hexadecyl thioester as revealed by gas chromatography-mass spectrometry.