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The genus Acinetobacter comprises both environmental and clinically relevant species associated with hospital-acquired infections. Among them, Acinetobacter baumannii is a critical priority bacterial pathogen, for which the research and development of new strategies for antimicrobial treatment are urgently needed. Acinetobacter spp. produce a variety of structurally diverse capsular polysaccharides (CPSs), which surround the bacterial cells with a thick protective layer. These surface structures are primary receptors for capsule-specific bacteriophages, that is, phages carrying tailspikes with CPS-depolymerizing/modifying activities. Phage tailspike proteins (TSPs) exhibit hydrolase, lyase, or esterase activities toward the corresponding CPSs of a certain structure. In this study, the data on all lytic capsule-specific phages infecting Acinetobacter spp. with genomes deposited in the NCBI GenBank database by January 2024 were summarized. Among the 149 identified TSPs encoded in the genomes of 143 phages, the capsular specificity (K specificity) of 46 proteins has been experimentally determined or predicted previously. The specificity of 63 TSPs toward CPSs, produced by various Acinetobacter K types, was predicted in this study using a bioinformatic analysis. A comprehensive phylogenetic analysis confirmed the prediction and revealed the possibility of the genetic exchange of gene regions corresponding to the CPS-recognizing/degrading parts of different TSPs between morphologically and taxonomically distant groups of capsule-specific Acinetobacter phages.

β-lactamases represent the main mechanism of antimicrobial resistance in Gram-negative pathogens. Their catalytic function (cleaving β-lactam antibiotics) occurs in the bacterial periplasm, where they are commonly reported as soluble proteins. A bioinformatic analysis reveals a significant number of putative lipidated β-lactamases, expected to be attached to the outer bacterial membrane. Notably, 60% of Class D OXA β-lactamases (all from Acinetobacter spp.) are predicted as membrane-anchored proteins. We demonstrate that two clinically relevant carbapenemases, OXA-23 and OXA-24/40, are membrane-bound proteins in A. baumannii . This cellular localization favors the secretion of these enzymes into outer membrane vesicles that transport them outside the boundaries of the cell. β-lactamase-loaded vesicles can protect populations of antibiotic-susceptible bacteria, enabling them to thrive in the presence of β-lactam antibiotics. The ubiquity of this phenomenon suggests that it may have influenced the dissemination of resistance mediated by Acinetobacter spp., particularly in polymicrobial infections, being a potent evolutionary advantage.

The emergence and spread of antibiotic resistance among Acinetobacter spp. have been investigated extensively. Most studies focused on the multiple antibiotic resistance genes located on plasmids or genomic resistance islands. On the other hand, the mechanisms controlling intrinsic resistance are still not well understood. In this study, we identified the novel subclass of aminoglycoside nucleotidyltransferase ANT(3\")-II in Acinetobacter spp., which comprised numerous variants distributed among three main clades. All members of this subclass can inactivate streptomycin and spectinomycin. The three ant(3\")-II genes, encoding for the three ANT(3\")-II clades, are widely distributed in the genus Acinetobacter and always located in the same conserved genomic region. According to their prevalence, these genes are intrinsic in Acinetobacter baumannii, Acinetobacter pittii, and Acinetobacter gyllenbergii. We also demonstrated that the ant(3\")-II genes are located in a homologous recombination hotspot and were recurrently transferred among Acinetobacter species. In conclusion, our findings demonstrated a novel mechanism of natural resistance in Acinetobacter spp., identified a novel subclass of aminoglycoside nucleotidyltransferase and provided new insight into the evolutionary history of intrinsic resistance genes.

Carbapenem-resistant Acinetobacter spp. is associated with nosocomial infections in intensive care unit patients, resulting in high mortality. Although Acinetobacter spp. represent a serious public health problem worldwide, there are a few studies related to the presence of carbapenemases in health care facilities and other environmental settings in Ecuador. The main aim of this study was to characterize the carbapenem-resistant Acinetobacter spp. isolates obtained from four hospitals (52) and from five rivers (27) close to Quito. We used the disc diffusion and EDTA sinergy tests to determine the antimicrobial susceptibility and the production of metallo β-lactamases, respectively. We carried out a multiplex PCR of gyrB gene and the sequencing of partial rpoB gene to bacterial species identification. We performed molecular screening of nine carbapenem-resistant genes ( bla SPM , bla SIM , bla GIM , bla GES , bla OXA-23 , bla OXA-24 , bla OXA-51 , bla OXA-58 , and bla OXA-143 ) by multiplex PCR, followed by identification using sequencing of bla OXA genes. Our findings showed that carbapenem-resistant A. baumannii were the main species found in health care facilities and rivers. Most of the clinical isolates came from respiratory tract samples and harbored bla OXA-23 , bla OXA-366 , bla OXA-72 , bla OXA-65 , bla OXA-70 , and bla OXA-143-like genes. The river isolates harbored only the bla OXA-51 and probably bla OXA-259 genes. We concluded that the most predominant type of carbapenem genes among isolates were both bla OXA-23 and bla OXA-65 among A. baumannii clinical isolates.

Acinetobacter baumannii is an opportunistic Gram-negative pathogen that causes a wide range of infections including pneumonia, septicemia, necrotizing fasciitis and severe wound and urinary tract infections. Analysis of A. baumannii representative strains grown in Chelex 100-treated medium for hemolytic activity demonstrated that this pathogen is increasingly hemolytic to sheep, human and horse erythrocytes, which interestingly contain increasing amounts of phosphatidylcholine in their membranes. Bioinformatic, genetic and functional analyses of 19 A. baumannii isolates showed that the genomes of each strain contained two phosphatidylcholine-specific phospholipase C (PC-PLC) genes, which were named plc1 and plc2. Accordingly, all of these strains were significantly hemolytic to horse erythrocytes and their culture supernatants tested positive for PC-PLC activity. Further analyses showed that the transcriptional expression of plc1 and plc2 and the production of phospholipase and thus hemolytic activity increased when bacteria were cultured under iron-chelation as compared to iron-rich conditions. Testing of the A. baumannii ATCC 19606T plc1::aph-FRT and plc2::aph isogenic insertion derivatives showed that these mutants had a significantly reduced PC-PLC activity as compared to the parental strain, while testing of plc1::ermAM/plc2::aph demonstrated that this double PC-PLC isogenic mutant expressed significantly reduced cytolytic and hemolytic activity. Interestingly, only plc1 was shown to contribute significantly to A. baumannii virulence using the Galleria mellonella infection model. Taken together, our data demonstrate that both PLC1 and PLC2, which have diverged from a common ancestor, play a concerted role in hemolytic and cytolytic activities; although PLC1 seems to play a more critical role in the virulence of A. baumannii when tested in an invertebrate model. These activities would provide access to intracellular iron stores this pathogen could use during growth in the infected host.

Acinetobacter baumannii is an important cause of multidrug-resistant hospital acquired infections in the world. Here, we investigate the presence of NDM-1 and other carbapenemases among carbapenem-resistant A. baumannii isolated between August 2010 and December 2014 from three large hospitals in Hanoi, Vietnam. We identified 23/582 isolates (4 %) (11 from hospital A, five from hospital B, and seven from hospital C) that were NDM-1 positive, and among them 18 carried additional carbapenemase genes, including seven isolates carrying NDM-1, IMP-1, and OXA-58 with high MICs for carbapenems. Genotyping indicated that NDM-1 carrying A. baumannii have expanded clonally in these hospitals. Five new STs (ST1135, ST1136, ST1137, ST1138, and ST1139) were identified. One isolate carried NDM-1 on a plasmid belonging to the N-repA replicon type; no NDM-1-positive plasmids were identified in the other isolates. We have shown the extent of the carbapenem resistance and the local clonal spread of A. baumannii carrying NDM-1 in these hospitals; coexistence of NDM-1 and IMP-1 is reported for the first time from Vietnam here, and this will further seriously limit future therapeutic options.

Acinetobacter species and Pseudomonas aeruginosa are noted for their intrinsic resistance to antibiotics and for their ability to acquire genes encoding resistance determinants. Foremost among the mechanisms of resistance in both of these pathogens is the production of β-lactamases and aminoglycoside-modifying enzymes. Additionally, diminished expression of outer membrane proteins, mutations in topoisomerases, and up-regulation of efflux pumps play an important part in antibiotic resistance. Unfortunately, the accumulation of multiple mechanisms of resistance leads to the development of multiply resistant or even “panresistant” strains.

Background Lignin holds great potential as an abundant and sustainable source of aromatic compounds, offering a viable alternative to fossil-based resources for producing chemicals and materials. Biological upgrading of lignin-derived aromatics can lead to more comprehensive lignocellulose utilization, thereby enhancing the overall feasibility of production. However, exploring a broader range of potential microbial hosts, pathways, and enzymes is crucial for developing efficient conversion processes. In particular, improving the conversion of S-lignin-related aromatics, such as syringate, remains a key area for future research. Results In this study, we aimed to investigate the conversion of S-lignin-related syringate in Acinetobacter baylyi ADP1 by exploiting its native vanillate demethylase, VanAB. We discovered that the wild-type strain can efficiently O -demethylate syringate to 3- O -methylgallate (3MGA) and then to gallate, revealing a previously unknown activity of VanAB of A. baylyi ADP1. Conversion dynamics and in vitro characterization showed that VanAB prefers syringate as a substrate over 3MGA. Overexpression of vanAB resulted in simultaneous conversion of syringate and 3MGA, but negatively impacted growth, potentially due to toxic side product formaldehyde and redox imbalance caused by high NADH consumption of the O -demethylation reactions. Native vanAB expression resulted in 3MGA accumulation if syringate was available. We took advantage of this by constructing a strain with heterologous expression of galA , a gallate dioxygenase from Pseudomonas putida KT2440, and demonstrated the conversion of the intermediate 3MGA into 2-pyrone-4,6,-dicarboxylate (PDC), a precursor for high-quality polyesters. Conclusions In this study, we discovered a previously unknown activity of syringate conversion in A. baylyi ADP1. By adjusting the expression level of vanAB , syringate can be directed either into gallate or 3MGA, which could be further converted into PDC through the heterologous expression of galA. Our results further highlight the potential and versatility of A. baylyi ADP1 for the conversion and upgrading of lignin-related aromatic compounds.

Among the Acinetobacter genus, Acinetobacter pittii stands out as an important opportunistic infection causative agent commonly found in hospital settings, which poses a serious threat to human health. Recently, the high prevalence of carbapenem-resistant A. pittii isolates has created significant therapeutic challenges for clinicians. Bacteriophages and their derived enzymes are promising therapeutic alternatives or adjuncts to antibiotics effective against multidrug-resistant bacterial infections. However, studies investigating the depolymerases specific to A. pittii strains are scarce. In this study, we identified and characterized a capsule depolymerase, Dpo27, encoded by the bacteriophage IME-Ap7, which targets A. pittii . A total of 23 clinical isolates of Acinetobacter spp. were identified as A. pittii (21.91%, 23/105), and seven A. pittii strains with various K locus (KL) types (KL14, KL32, KL38, KL111, KL163, KL207, and KL220) were used as host bacteria for phage screening. The lytic phage IME-Ap7 was isolated using A. pittii 7 (KL220) as an indicator bacterium and was observed for depolymerase activity. A putative tail fiber gene encoding a polysaccharide-degrading enzyme (Dpo27) was identified and expressed. The results of the modified single-spot assay showed that both A. pittii 7 and 1492 were sensitive to Dpo27, which was assigned the KL220 type. After incubation with Dpo27, A. pittii strain was susceptible to killing by human serum; moreover, the protein displayed no hemolytic activity against erythrocytes. Furthermore, the protein exhibited sustained activity across a wide pH range (5.0–10.0) and at temperatures between 20 and 50°C. In summary, the identified capsule depolymerase Dpo27 holds promise as an alternative treatment for combating KL220-type A. pittii infections.

Microbes have already been engineered to produce diesel fuels, and now the microbial production of components of petrol (gasoline) including short-chain alkanes has been achieved using Escherichia coli strains metabolically engineered with components of fatty acid biosynthesis pathways. Engineering bacteria to pump gasoline High oil prices and the depletion of fossil resources have fuelled extensive research on the production of sustainable biofuels from renewable resources. Engineered microbes are one option, but until now microbes have not produced gasoline, a mixture of lighter liquid hydrocarbons in the range C4 to C12, in part because cellular metabolism favours the production of mainly long-chain fatty acids and their derivatives. Here Yong Jun Choi and Sang Yup Lee describe Escherichia coli strains engineered to produce short-chain alkanes, free fatty acids, fatty esters and fatty alcohols. The final engineered strain produced as much as 580.8 milligrams per litre of short-chain alkanes, primarily nonane and decane. The metabolic engineering strategies described here should be useful in designing microorganisms for the production of short-chain fatty acids and derivatives as many useful industrial fuels and chemicals. Increasing concerns about limited fossil fuels and global environmental problems have focused attention on the need to develop sustainable biofuels from renewable resources. Although microbial production of diesel has been reported, production of another much in demand transport fuel, petrol (gasoline), has not yet been demonstrated. Here we report the development of platform Escherichia coli strains that are capable of producing short-chain alkanes (SCAs; petrol), free fatty acids (FFAs), fatty esters and fatty alcohols through the fatty acyl (acyl carrier protein (ACP)) to fatty acid to fatty acyl-CoA pathway. First, the β-oxidation pathway was blocked by deleting the fadE gene to prevent the degradation of fatty acyl-CoAs generated in vivo . To increase the formation of short-chain fatty acids suitable for subsequent conversion to SCAs in vivo , the activity of 3-oxoacyl-ACP synthase (FabH) 1 , which is inhibited by unsaturated fatty acyl-ACPs 2 , was enhanced to promote the initiation of fatty acid biosynthesis by deleting the fadR gene; deletion of the fadR gene prevents upregulation of the fabA and fabB genes responsible for unsaturated fatty acids biosynthesis 3 . A modified thioesterase 4 was used to convert short-chain fatty acyl-ACPs to the corresponding FFAs, which were then converted to SCAs by the sequential reactions of E. coli fatty acyl-CoA synthetase, Clostridium acetobutylicum fatty acyl-CoA reductase and Arabidopsis thaliana fatty aldehyde decarbonylase. The final engineered strain produced up to 580.8 mg l −1 of SCAs consisting of nonane (327.8 mg l −1 ), dodecane (136.5 mg l −1 ), tridecane (64.8 mg l −1 ), 2-methyl-dodecane (42.8 mg l −1 ) and tetradecane (8.9 mg l −1 ), together with small amounts of other hydrocarbons. Furthermore, this platform strain could produce short-chain FFAs using a fadD -deleted strain, and short-chain fatty esters by introducing the Acinetobacter sp. ADP1 wax ester synthase ( atfA ) 5 and the E. coli mutant alcohol dehydrogenase ( adhE mut ) 6 .