Archaic chaperone-usher pili self-secrete into superelastic zigzag springs.

Adhesive pili assembled through the chaperone-usher pathway are hair-like appendages that mediate host tissue colonization and biofilm formation of Gram-negative bacteria1-3. Archaic chaperone-usher pathway pili, the most diverse and widespread chaperone-usher pathway adhesins, are promising vaccine and drug targets owing to their prevalence in the most troublesome multidrug-resistant pathogens1,4,5. However, their architecture and assembly-secretion process remain unknown. Here, we present the cryo-electron microscopy structure of the prototypical archaic Csu pilus that mediates biofilm formation of Acinetobacter baumannii-a notorious multidrug-resistant nosocomial pathogen. In contrast to the thick helical tubes of the classical type 1 and P pili, archaic pili assemble into an ultrathin zigzag architecture secured by an elegant clinch mechanism. The molecular clinch provides the pilus with high mechanical stability as well as superelasticity, a property observed for the first time, to our knowledge, in biomolecules, while enabling a more economical and faster pilus production. Furthermore, we demonstrate that clinch formation at the cell surface drives pilus secretion through the outer membrane. These findings suggest that clinch-formation inhibitors might represent a new strategy to fight multidrug-resistant bacterial infections.

The Wzi outer membrane protein mediates assembly of a tight capsular polysaccharide layer on the Acinetobacter baumannii cell surface.

Identification of novel therapeutic targets is required for developing alternate strategies to treat infections caused by the extensively drug-resistant bacterial pathogen, Acinetobacter baumannii. As capsular polysaccharide (CPS) is a prime virulence determinant required for evasion of host immune defenses, understanding the pathways for synthesis and assembly of this discrete cell-surface barrier is important. In this study, we assess cell-bound and cell-free CPS material from A. baumannii AB5075 wildtype and transposon library mutants and demonstrate that the Wzi outer membrane protein is required for the proper assembly of the CPS layer on the cell surface. Loss of Wzi resulted in an estimated 4.4-fold reduction in cell-associated CPS with a reciprocal increase in CPS material shed in the extracellular surrounds. Transmission electron microscopy revealed a disrupted CPS layer with sparse patches of CPS on the external face of the outer membrane when Wzi function was lost. However, this genotype did not have a significant effect on biofilm formation. Genetic analysis demonstrated that the wzi gene is ubiquitous in the species, though the nucleotide sequences were surprisingly diverse. Though divergence was not concomitant with variation at the CPS biosynthesis K locus, an association between wzi type and the first sugar of the CPS representing the base of the structure most likely to interact with Wzi was observed.

Comparison of the Phytochemical Composition and Antibacterial Activities of the Various Extracts from Leaves and Twigs of Illicium verum.

Previous studies have revealed the numerous biological activities of the fruits of Illicium verum; however, the activities of its leaves and twigs have remained undiscovered. The study aimed to investigate the phytochemical components and antibacterial activity of the various extracts from the leaves and twigs of Illicium verum. The herbal extracts were prepared by supercritical CO2 extraction (SFE) and 95% ethanol extraction, followed by partition extraction based on solvent polarity. Analysis of antimicrobial activity was conducted through the usage of nine clinical antibiotic- resistant isolates, including Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii. Among the tested samples, the SFE extracts exhibited broader and stronger antibacterial activities against the test strains, with a range of MIC between 0.1-4.0 mg/mL and MBC between 0.2-4.5 mg/mL. Observations made through scanning electron microscopy revealed potential mechanism of the antimicrobial activities involved disruption of membrane integrity of the test pathogens. Evaluation of the chemical composition by gas chromatography-mass spectrometry indicated the presence of anethole, anisyl aldehyde, anisyl acetone and anisyl alcohol within the SFE extracts, demonstrating significant correlations with the antibacterial activities observed. Therefore, the leaves and twigs of Illicium verum hold great potential in being developed as new natural antibacterial agents.

Cryo-EM Determination of Eravacycline-Bound Structures of the Ribosome and the Multidrug Efflux Pump AdeJ of Acinetobacter baumannii.

Antibiotic-resistant strains of the Gram-negative pathogen Acinetobacter baumannii have emerged as a significant global health threat. One successful therapeutic option to treat bacterial infections has been to target the bacterial ribosome. However, in many cases, multidrug efflux pumps within the bacterium recognize and extrude these clinically important antibiotics designed to inhibit the protein synthesis function of the bacterial ribosome. Thus, multidrug efflux within A. baumannii and other highly drug-resistant strains is a major cause of failure of drug-based treatments of infectious diseases. We here report the first structures of the Acinetobacter drug efflux (Ade)J pump in the presence of the antibiotic eravacycline, using single-particle cryo-electron microscopy (cryo-EM). We also describe cryo-EM structures of the eravacycline-bound forms of the A. baumannii ribosome, including the 70S, 50S, and 30S forms. Our data indicate that the AdeJ pump primarily uses hydrophobic interactions to bind eravacycline, while the 70S ribosome utilizes electrostatic interactions to bind this drug. Our work here highlights how an antibiotic can bind multiple bacterial targets through different mechanisms and potentially enables drug optimization by taking advantage of these different modes of ligand binding. IMPORTANCE Acinetobacter baumannii has developed into a highly antibiotic-resistant Gram-negative pathogen. The prevalent AdeJ multidrug efflux pump mediates resistance to different classes of antibiotics known to inhibit the function of the 70S ribosome. Here, we report the first structures of the A. baumannii AdeJ pump, both in the absence and presence of eravacycline. We also describe structures of the A. baumannii ribosome bound by this antibiotic. Our results indicate that AdeJ and the ribosome use very distinct binding modes for drug recognition. Our work will ultimately enable structure-based drug discovery to combat antibiotic-resistant A. baumannii infection.

Accelerated antimicrobial discovery via deep generative models and molecular dynamics simulations.

The de novo design of antimicrobial therapeutics involves the exploration of a vast chemical repertoire to find compounds with broad-spectrum potency and low toxicity. Here, we report an efficient computational method for the generation of antimicrobials with desired attributes. The method leverages guidance from classifiers trained on an informative latent space of molecules modelled using a deep generative autoencoder, and screens the generated molecules using deep-learning classifiers as well as physicochemical features derived from high-throughput molecular dynamics simulations. Within 48 days, we identified, synthesized and experimentally tested 20 candidate antimicrobial peptides, of which two displayed high potency against diverse Gram-positive and Gram-negative pathogens (including multidrug-resistant Klebsiella pneumoniae) and a low propensity to induce drug resistance in Escherichia coli. Both peptides have low toxicity, as validated in vitro and in mice. We also show using live-cell confocal imaging that the bactericidal mode of action of the peptides involves the formation of membrane pores. The combination of deep learning and molecular dynamics may accelerate the discovery of potent and selective broad-spectrum antimicrobials.

Cryoelectron Microscopy Structures of AdeB Illuminate Mechanisms of Simultaneous Binding and Exporting of Substrates.

Acinetobacter baumannii is a Gram-negative pathogen that has emerged as one of the most highly antibiotic-resistant bacteria worldwide. Multidrug efflux within these highly drug-resistant strains and other opportunistic pathogens is a major cause of failure of drug-based treatments of infectious diseases. The best-characterized multidrug efflux system in A. baumannii is the prevalent Acinetobacterdrug efflux B (AdeB) pump, which is a member of the resistance-nodulation-cell division (RND) superfamily. Here, we report six structures of the trimeric AdeB multidrug efflux pump in the presence of ethidium bromide using single-particle cryoelectron microscopy (cryo-EM). These structures allow us to directly observe various novel conformational states of the AdeB trimer, including the transmembrane region of trimeric AdeB can be associated with form a trimer assembly or dissociated into "dimer plus monomer" and "monomer plus monomer plus monomer" configurations. We also discover that a single AdeB protomer can simultaneously anchor a number of ethidium ligands and that different AdeB protomers can bind ethidium molecules simultaneously. Combined with molecular dynamics (MD) simulations, we reveal a drug transport mechanism that involves multiple multidrug-binding sites and various transient states of the AdeB membrane protein. Our data suggest that each AdeB protomer within the trimer binds and exports drugs independently.IMPORTANCEAcinetobacter baumannii has emerged as one of the most highly antibiotic-resistant Gram-negative pathogens. The prevalent AdeB multidrug efflux pump mediates resistance to a broad spectrum of clinically relevant antimicrobial agents. Here, we report six cryo-EM structures of the trimeric AdeB pump in the presence of ethidium bromide. We discover that a single AdeB protomer can simultaneously anchor a number of ligands, and different AdeB protomers can bind ethidium molecules simultaneously. The results indicate that each AdeB protomer within the trimer recognizes and extrudes drugs independently.

Acquired mucoid phenotype of Acinetobacter baumannii: Impact for the molecular characteristics and virulence.

Mucoid phenotype is an important adaptive defense response for Acinetobacter baumannii (A. baumannii). The aim of this study was to analyze the impact of mucoid phenotype for the molecular characteristics and virulence of A. baumannii. We observed that the colonies of mucoid A. baumannii were moist, with an elevated surface, and the wire drawing result was positive. Transmission electron microscopy data showed that the outer wall of the mucoid colonies was not smooth, had protruding pseudopodia, and was surrounded by a layer of unknown material. Antibiotic susceptibility testing showed that the mucoid strains were multidrug resistant. Notably, the mucoid phenotype and antibiotic resistance were not correlated with the amount of biofilm produced by A. baumannii. MLST data demonstrated that the mucoid A. baumannii strains belonged to type ST2. Most (82.6 %, 38/46) of the multidrug-resistant nonmucoid strains also belonged to the molecular type ST2 and to other types, including ST129, ST158, ST195, ST80 and ST3. Moreover, mucoid A. baumannii strains were more virulent than nonmucoid isolates in a mouse model. The comparative transcriptomic data indicated that 15 genes, especially IX87_RS16955 (acnA), IX87_RS10800 (XanP), IX87_RS12875 (GlmM), IX87_RS00885 and IX87_RS12395 (bfr), were possibly associated with the phenotype and virulence of mucoid A. baumannii. In conclusions, the study comprehensively describes the molecular characteristics and virulence regulatory mechanism of mucoid A. baumannii, and provides novel insights for the prevention and treatment of infections associated with these strains.

Development of Different Methods for Preparing Acinetobacter baumannii Outer Membrane Vesicles Vaccine: Impact of Preparation Method on Protective Efficacy.

Acinetobacter baumannii (A. baumannii) is becoming a common global concern due to the emergence of multi-drug or pan-drug resistant strains. Confronting the issue of antimicrobial resistance by developing vaccines against the resistant pathogen is becoming a common strategy. In this study, different methods for preparing A. baumannii outer membrane vesicles (AbOMVs) vaccines were developed. sOMV (spontaneously released AbOMV) was extracted from the culture supernatant, while SuOMV (sucrose-extracted AbOMV) and nOMV (native AbOMV) were prepared from the bacterial cells. Three AbOMVs exhibited significant differences in yield, particle size, protein composition, and LPS/DNA content. To compare the protective efficacy of the three AbOMVs, groups of mice were immunized either intramuscularly or intranasally with each AbOMV. Vaccination via both routes conferred significant protection against lethal and sub-lethal A. baumannii challenge. Moreover, intranasal vaccination provided more robust protection, which may be attributed to the induction of significant sIgA response in mucosal sites. Among the three AbOMVs, SuOMV elicited the highest level of protective immunity against A. baumannii infection, whether intramuscular or intranasal immunization, which was characterized by the expression of the most profound specific serum IgG or mucosal sIgA. Taken together, the preparation method had a significant effect on the yield, morphology, and composition of AbOMVs, that further influenced the protective effect against A. baumannii infection.

Cryo-electron Microscopy Structure of the Acinetobacter baumannii 70S Ribosome and Implications for New Antibiotic Development.

Antimicrobial resistance is a major health threat as it limits treatment options for infection. At the forefront of this serious issue is Acinetobacter baumannii, a Gram-negative opportunistic pathogen that exhibits the remarkable ability to resist antibiotics through multiple mechanisms. As bacterial ribosomes represent a target for multiple distinct classes of existing antimicrobial agents, we here use single-particle cryo-electron microscopy (cryo-EM) to elucidate five different structural states of the A. baumannii ribosome, including the 70S, 50S, and 30S forms. We also determined interparticle motions of the 70S ribosome in different tRNA bound states using three-dimensional (3D) variability analysis. Together, our structural data further our understanding of the ribosome from A. baumannii and other Gram-negative pathogens and will enable structure-based drug discovery to combat antibiotic-resistant bacterial infections.IMPORTANCEAcinetobacter baumannii is a severe nosocomial threat largely due to its intrinsic antibiotic resistance and remarkable ability to acquire new resistance determinants. The bacterial ribosome serves as a major target for modern antibiotics and the design of new therapeutics. Here, we present cryo-EM structures of the A. baumannii 70S ribosome, revealing several unique species-specific structural features that may facilitate future drug development to combat this recalcitrant bacterial pathogen.

Ω76: A designed antimicrobial peptide to combat carbapenem- and tigecycline-resistant Acinetobacter baumannii.

Drug resistance is a public health concern that threatens to undermine decades of medical progress. ESKAPE pathogens cause most nosocomial infections, and are frequently resistant to carbapenem antibiotics, usually leaving tigecycline and colistin as the last treatment options. However, increasing tigecycline resistance and colistin's nephrotoxicity severely restrict use of these antibiotics. We have designed antimicrobial peptides using a maximum common subgraph approach. Our best peptide (Ω76) displayed high efficacy against carbapenem and tigecycline-resistant Acinetobacter baumannii in mice. Mice treated with repeated sublethal doses of Ω76 displayed no signs of chronic toxicity. Sublethal Ω76 doses co-administered alongside sublethal colistin doses displayed no additive toxicity. These results indicate that Ω76 can potentially supplement or replace colistin, especially where nephrotoxicity is a concern. To our knowledge, no other existing antibiotics occupy this clinical niche. Mechanistically, Ω76 adopts an α-helical structure in membranes, causing rapid membrane disruption, leakage, and bacterial death.

Synergistic antibacterial effect of ultrasound microbubbles combined with chitosan-modified polymyxin B-loaded liposomes on biofilm-producing Acinetobacter baumannii.

PURPOSE: Resistant strains of Acinetobacter baumannii (AB) that can form biofilms are resistant to polymyxin. Therefore, effective and safe polymyxin preparations against biofilm-producing AB are urgently needed. This study aims to prepare chitosan-modified polymyxin B-loaded liposomes (CLPs) and ultrasound microbubbles (USMBs) and then explore the synergistic antibacterial effects of USMBs combined with CLPs in vitro. METHODS: CLPs were prepared using a modified injection method, and microbubbles were prepared using a simple mechanical vibration method. Minimal biofilm inhibitory concentration (MBIC) of CLPs against resistant biofilm-producing AB was determined. Antibacterial activities of CLPs with or without USMBs were analyzed by crystal violet staining and resazurin assays to evaluate biofilm mass and viable counts, respectively. Then, the anti-biofilm effects of CLPs with or without USMBs on biofilm-producing AB were confirmed via scanning electron microscopy (SEM) analysis. RESULTS: We prepared CLPs that were 225.17±17.85 nm in size and carried positive charges of 12.64±1.44 mV. These CLPs, with higher encapsulation efficiency and drug loading, could exhibit a sustained release effect. We prepared microbubbles that were 2.391±0.052 µm in size and carried negative charges of -4.32±0.43 mV. The MBICs of the CLPs on the biofilm-producing AB was 8±2 µg/mL, while that of polymyxin B was 32±2 µg/mL. USMBs in combination with 2 µg/mL of polymyxin B could completely eliminate the biofilm-producing AB and achieve the maximum antimicrobial effects (P>0.05 vs sterile blank control). SEM imaging revealed some scattered bacteria without a biofilm structure in the USMB combined with the CLP group, confirming that this combination has the greatest anti-biofilm effects. CONCLUSION: In this research, we successfully prepared USMBs and CLPs that have a more significant antibacterial effect on biofilm-forming AB than polymyxin B alone. Experiments in vitro indicate that the synergistic antibacterial effect of combining USMBs with CLPs containing as little as 2 µg/mL of polymyxin B is sufficient to almost eliminate drug-resistant biofilm-producing AB.

Eradication of persister cells of Acinetobacter baumannii through combination of colistin and amikacin antibiotics.

OBJECTIVES: Persister cells following antibiotic exposure may cause failure of antibiotic treatment. The synergistic effects of antibiotic combinations with respect to eliminating persister cells were investigated based on their characteristics. METHODS: For Acinetobacter baumannii clinical isolates, persister assays were performed using colistin, amikacin, imipenem and ciprofloxacin in various ways, including exposure to antibiotics in combination and sequentially. Persister phenotypes were observed through analysis of ATP concentration, membrane potential and transmission electron microscopy. RESULTS: Each A. baumannii isolate showed a specific survival rate of persister cells against each antibiotic. The persister cells were eradicated effectively by exposure to the combination of colistin and amikacin, especially in the sequential order of colistin then amikacin. While the persister cells were not identified after 6 h when exposed to the antibiotics in the order colistin then amikacin, they remained at 0.016% when antibiotic exposure was done in the order amikacin then colistin. Although membrane potential was low in both colistin and amikacin persisters, depletion of the intracellular ATP concentration was only observed in colistin persisters. In addition, transmission electron microscopy analysis showed that colistin persisters have a unique morphology with a rough and rippled membrane and many outer membrane vesicles. Empty pore-like structures surrounded by cracks were also observed. CONCLUSIONS: In A. baumannii, the combination of colistin and amikacin was most effective for eradication of persister cells, probably due to different mechanisms of persister cell formation between antibiotics. It was also identified that the sequential order of colistin followed by amikacin was important to eradicate the persister cells.

Skin and Soft Tissue Models for Acinetobacter baumannii Infection.

Multidrug-resistant A. baumannii are important Gram-negative pathogens causing persistent wound infections in both wounded and burned victims, which often result in secondary complications such as delayed wound healing, skin graft failure, and sometimes more serious outcomes such as sepsis and amputation. The choice of antibiotics to remediate these A. baumannii infections is becoming limited; and therefore, there has been a renewed interest in the research and development of new antibacterials targeting this pathogen. However, the evaluation of safety and efficacy is made more difficult by the lack of well-established in vivo models. This chapter describes established rodent and large animal models that have been used to investigate and develop treatments for A. baumannii skin and soft tissue infections.

Biotic and abiotic substrates for enhancing Acinetobacter baumannii biofilm formation: New approach using extracellular matrix and slanted coverslip technique.

Acinetobacter baumannii has been well recognized as a problematic human pathogen and several reports has shown the incidence of multidrug and pandrug-resistant A. baumannii strains in infirmary infections. A. baumannii grows only on an air-liquid interface and does not form a contiguous biofilm. Extracellular matrices (ECM) and slanted glass coverslips are (SGC) used as biofilm substrates and biofilms have been investigated by SEM, confocal and crystal violet staining. ECM has shown enhanced biofilm formation under dynamic conditions rather than static conditions. SGC biofilm yield assay has shown higher levels of continuous layers and packed thicker biofilm formation with glass coverslip inserts, up to 1.7 to 3 times higher biofilm formation, than when compared with no glass coverslip inserts. A media immersed ECM study revealed that biofilm grown on extracellular matrixes formed thread-like pili structures, and that these structures had contact with the ECM and also showed excellent cell-to-cell interaction. In summary, A. baumannii showed higher biofilm formation capacities with ECM, while the prominent results were directly related with the biofilm formation capacity of A. baumannii. For the initial step of biofilm formation, adherence is an important factor and, consequently, strains with a comparatively high capability to adhere to extracellular matrices and slanted glass coverslips provide a new method of enhanced biofilm growth for in vitro assays. ECM can be used as a substrate for immersed biofilm formation studies and the SGC method for air-liquid interface exposed biofilm formation studies, and these substrates can provide better biofilm growth and easy handling for in vitro adherence and biofilm assays.

Efficacy of designer K11 antimicrobial peptide (a hybrid of melittin, cecropin A1 and magainin 2) against Acinetobacter baumannii-infected wounds.

Due to emergence of multidrug resistance in pathogens, the attention of the scientific community is now directed towards strengthening the reservoir of antimicrobial compounds. Prior to in vivo studies, the interaction and penetration of a hybrid peptide K11 in bacterial cells using confocal microscopy was assessed which was observed as early as 10 min after incubation with the peptide. Cell lysis along with leakage of cytoplasmic content was confirmed by electron microscopy. To evaluate the in vivo performance of the peptide, it was contained in carbopol hydrogel. Efficacy of the hydrogel formulation was then evaluated against Acinetobacter baumannii-infected wounds using a murine excision model. Treatment resulted in restoration of body weight, complete clearance of infection from the wound by day 7 and 99% wound enclosure by day 21, in contrast to the persistence of infection and 70% wound enclosure in the infected group. Further, this treatment resulted in a 2.6-fold decrease in the levels of malondialdehyde along with a 4.5-fold increase in the levels of catalase on day 3. Appearance of normal histo-architecture was observed in the treatment group. Based on these results, the peptide hydrogel can be exploited in future as one of the strategies for developing a topical anti-infective therapeutic agent.

Acinetobacter baumannii maintains its virulence after long-time starvation.

Acinetobacter baumannii is a cause of healthcare-associated infections. Although A. baumannii is an opportunistic pathogen, its infections are notoriously difficult to treat due to intrinsic and acquired antimicrobial resistance, often limiting effective therapeutic options. A. baumannii can survive for long periods in the hospital environment, particularly on inanimate surfaces. Such environments may act as a reservoir for cross-colonization and infection outbreaks and should be considered a substantial factor in infection control practices. Moreover, clothing of healthcare personnel and gadgets may play a role in the spread of nosocomial bacteria. A link between contamination of hospital surfaces and A. baumannii infections or between its persistence in the environment and its virulence has not yet been established. Bacteria under stress (i.e., long-term desiccation in hospital setting) could conserve factors that favor infection. To investigate whether desiccation and/or starvation may be involved in the ability of certain strains of A. baumannii to retain virulence factors, we have studied five well-characterized clinical isolates of A. baumannii for which survival times were determined under simulated hospital conditions. Despite a considerable reduction in the culturability over time (up to 88% depending on strain and the condition tested), some A. baumannii strains were able to maintain their ability to form biofilms after rehydration, addition of nutrients, and changing temperature. Also, after long-term desiccation, several clinical strains were able to grow in the presence of non-immune human serum as fine as their non-stressed homologs. Furthermore, we also show that the ability of bacterial strains to kill Galleria mellonella larvae does not change although A. baumannii cells were stressed by long-term starvation (up to 60 days). This means that A. baumannii can undergo a rapid adaptation to both the temperature shift and nutrients availability, conditions that can be easily found by bacteria in a new patient in the hospital setting.

Biofilm inhibitory efficiency of phytol in combination with cefotaxime against nosocomial pathogen Acinetobacter baumannii.

AIMS: This study aimed to evaluate the antibiofilm potential of phytol and cefotaxime combinations (PCCs) against Acinetobacter baumannii and to elucidate the molecular mechanism of their antibiofilm potential through the transcriptomic approach. METHODS AND RESULTS: Phytol and cefotaxime combination(s) (PCC(s) [160 µg ml-1  + 8 µg ml-1 for microbial type culture collection (MTCC) strain and 160 µg ml-1  + 0.5 µg ml-1 for clinical isolate] effectively inhibited the A. baumannii biofilm formation. Additionally, light, confocal laser scanning and scanning electron microscopic analyses validated the antibiofilm potential of PCCs. Furthermore, PCCs treated A. baumannii cells showed a decreased level of hydrophobicity index compared to their respective controls. Fourier-transform infrared (FT-IR) spectra of exopolysaccharide matrix extracted from PCCs-treated A. baumannii cells showed a visible decrease in absorbance of polysaccharides, nucleic acids and protein regions compared to the spectra of untreated controls. In the blood sensitivity assay, the PCCs-treated A. baumannii plates showed reduced a number of bacterial colonies compared to their control plates. Reduced level of catalase production was also observed in the PCCs treatment compared to their controls. Transcriptomic analysis revealed the downregulation of bfmR, bap, csuA/B, ompA, pgaA, pgaC and katE biofilm virulence genes in both the A. baumannii strains on treatment with PCCs. CONCLUSION: The obtained results of this study indicate that PCCs have potent antibiofilm activity and downregulate the biofilm-related virulence genes expression in A. baumannii. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the pioneering study, which shows the antibiofilm effect of PCCs against A. baumannii along with their molecular mechanism. The antibiofilm effect of PCCs could be a successful strategy for eradicating infections related to A. baumannii biofilms in nosocomial settings.

Levels of persisters influenced by aeration in Acinetobacter calcoaceticus-baumannii.

AIM: To evaluate the influence of aeration on persister levels from Acinetobacter calcoaceticus-baumannii isolates exposed to meropenem or tobramycin, as well as analyze morphological and structural changes in persisters. MATERIALS & METHODS: Levels of persisters were determined after a 48-h exposure to tobramycin or meropenem under aerated or static conditions, and persisters were analyzed by scanning and transmission electron microscopy. RESULTS: The fractions of persisters varied between isolates. Aeration reduced cell survival under each antibiotic treatment, and cell survival decreased as the tobramycin concentration was increased. Interestingly, division septa were observed in persisters by electron microscopy. CONCLUSION: Aeration may have stimulated bacterial growth, providing more targets for antibiotic action and leading to increased production of reactive oxygen species, which decreased levels of persisters.

Highly potent antimicrobial modified peptides derived from the Acinetobacter baumannii phage endolysin LysAB2.

The increase in the prevalence of multidrug-resistant Acinetobacter baumannii (MDRAB) strains is a serious public health concern. Antimicrobial peptides (AMPs) are a possible solution to this problem. In this study, we examined whether AMPs could be derived from phage endolysins. We synthesized four AMPs based on an amphipathic helical region in the C-terminus of endolysin LysAB2 encoded by the A. baumannii phage ΦAB2. These peptides showed potent antibacterial activity against A. baumannii (minimum inhibitory concentration, 4-64 µM), including some MDR and colistin-resistant A. baumannii. Of the four peptides, LysAB2 P3, with modifications that increased its net positive charge and decreased its hydrophobicity, showed high antibacterial activity against A. baumannii but little haemolytic and no cytotoxic activity against normal eukaryotic cells. The results of electron microscopy experiments and a fluorescein isothiocyanate staining assay indicated that this peptide killed A. baumannii through membrane permeabilization. Moreover, in a mouse intraperitoneal infection model, at 4 h after the bacterial injection, LysAB2 P3 decreased the bacterial load by 13-fold in ascites and 27-fold in blood. Additionally, LysAB2 P3 rescued sixty percent of mice heavily infected with A. baumannii from lethal bacteremia. Our results confirmed that bacteriophage endolysins are a promising resource for developing effective AMPs.

In vitro study of virulence potential of Acinetobacter baumannii outer membrane vesicles.

Acinetobacter baumannii has emerged as an important opportunistic pathogen mostly causing nosocomial infections. The virulence factors of this important pathogen are largely unknown. Outer membrane vesicles (OMV) are naturally secreted by many gram negative and gram positive bacteria. These vesicles contain outer membrane proteins, lipids, periplasmic proteins, DNA and RNA. Their role in intracellular and intercellular signaling, transfer of virulence factors and eliciting immune response in host cells has been established in many pathogens. In this study, we investigated OMVs from three multi-drug resistant (MDR) clinical isolates and a non-MDR reference strain of A. baumannii for virulence potential. A. baumannii OMVs showed phospholipase C, hemolytic and leukotoxic activities. We found large variations in virulence potential between OMVs of MDR clinical isolates and non-MDR reference strain. These effector molecules were concentrated in OMVs than whole cell bacterial culture and cell-free supernatant. OMV-mediated phospholipase, hemolytic and leucotoxic activities may have a key role in pathogenicity of A. baumannii infection and may be future targets for therapeutic and preventive strategies. This is, to the best of our knowledge, the first report showing virulence potential of A. baumannii OMVs.