Baumannii Cells Research Articles

Bacteriophage and Phage-Encoded Depolymerase Exhibit Antibacterial Activity Against K9-Type Acinetobacter baumannii in Mouse Sepsis and Burn Skin Infection Models.

Acinetobacter baumannii is a widely distributed nosocomial pathogen that causes various acute and chronic infections, particularly in immunocompromised patients. In this study, the activities of the K9-specific virulent phage AM24 and phage-encoded depolymerase DepAPK09 were assessed using in vivo mouse sepsis and burn skin infection models. In the mouse sepsis model, in the case of prevention or early treatment, a single K9-specific phage or recombinant depolymerase injection was able to protect 100% of the mice after parenteral infection with a lethal dose of A. baumannii of the K9-type, with complete eradication of the pathogen. In the case of delayed treatment, mouse survival decreased to 70% when injected with the phage and to 40% when treated with the recombinant enzyme. In the mouse burn skin infection model, the number of A. baumannii cells on the surface of the wound and in the deep layers of the skin decreased by several-fold after treatment with both the K9-specific phage and the recombinant depolymerase. The phage and recombinant depolymerase were highly stable and retained activity under a wide range of temperatures and pH values. The results obtained contribute to expanding our understanding of the in vivo therapeutic potential of specific phages and phage-derived depolymerases interacting with A. baumannii of different capsular types.

OXA β-lactamases from Acinetobacter spp. are membrane bound and secreted into outer membrane vesicles.

β-lactamases from Gram-negative bacteria are generally regarded as soluble, periplasmic enzymes. NDMs have been exceptionally characterized as lipoproteins anchored to the outer membrane. A bioinformatics study on all sequenced β-lactamases was performed that revealed a predominance of putative lipidated enzymes in the Class D OXAs. Namely, 60% of the OXA Class D enzymes contain a lipobox sequence in their signal peptide, that is expected to trigger lipidation and membrane anchoring. This contrasts with β-lactamases from other classes, which are predicted to be mostly soluble proteins. Almost all (>99%) putative lipidated OXAs are present in Acinetobacter spp. Importantly, we further demonstrate that OXA-23 and OXA-24/40 are lipidated, membrane-bound proteins in Acinetobacter baumannii. In contrast, OXA-48 (commonly produced by Enterobacterales) lacks a lipobox and is a soluble protein. Outer membrane vesicles (OMVs) from A. baumannii cells expressing OXA-23 and OXA-24/40 contain these enzymes in their active form. Moreover, OXA-loaded OMVs were able to protect A. baumannii, Escherichia coli, and Pseudomonas aeruginosa cells susceptible to piperacillin and imipenem. These results permit us to conclude that membrane binding is a bacterial host-specific phenomenon in OXA enzymes. These findings reveal that membrane-bound β-lactamases are more common than expected and support the hypothesis that OMVs loaded with lipidated β-lactamases are vehicles for antimicrobial resistance and its dissemination. This advantage could be crucial in polymicrobial infections, in which Acinetobacter spp. are usually involved, and underscore the relevance of identifying the cellular localization of lactamases to better understand their physiology and target them.IMPORTANCEβ-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.

Unveiling the Self-assembly and Therapeutic Efficacy of Antimicrobial Peptides SA4 Against Multidrug-Resistant A. baumannii.

Infections linked to Acinetobacter baumannii are one of the main risks of modern medicine. Biofilms formed by A. baumannii due to a protective extracellular polysaccharide matrix make them highly tolerant to conventional antibiotics and raise the possibility of antibiotic resistance. Antimicrobial peptides (AMPs) are gaining popularity due to their broad-spectrum actions and key properties of peptide self-assembly, making them a promising alternative to antibiotics. Here, we demonstrate that 12-residue synthetic self-assembled peptide SA4 nanostructures have enough antibacterial action to prevent the growth of mature bacterial biofilms. The SA4 peptide was successfully synthesized by using the solid-phase peptide synthesis method, and its self-assembly was prepared in water. The self-assembled peptide hydrogel formed nanotube structure was observed under a scanning electron microscope and further characterized to confirm their physical and molecular properties. The resulting hydrogel exhibits significant antibacterial activity against MDR A. baumannii strains (MDR-1 and MDR-2), responsible for many nosocomial infections. In addition, at various gel concentrations, this hydrogel has the potential to inhibit about 30-80% of biofilms formed by MDR strains. Furthermore, under a microscope, it has been observed that the rupture of the bacterial cell membrane and cell wall of A. baumannii cells is caused by peptide nanotubes generated by self-assemblies. Thus, peptide-based nanotubes present intriguing avenues for various biomedical applications. This is the first report of bacterial biofilm removal with SA4 peptide nanotubes, and offering a unique treatment for infections linked to biofilms.

Experimental evolution under different nutritional conditions changes the genomic architecture and virulence of Acinetobacter baumannii

This study uncovers the molecular processes governing the adaptive evolution of multidrug-resistant (MDR) pathogens without antibiotic pressure. Genomic analysis of MDR Acinetobacter baumannii cells cultured for 8000 generations under starvation conditions (EAB1) or nutrient-rich conditions (EAB2) revealed significant genomic changes, primarily by insertion sequence (IS)-mediated insertions and deletions. Only two Acinetobacter-specific prophage-related deletions and translocations were observed in the EAB1 strain. Both evolved strains exhibited higher virulence in mouse infection studies, each with different modes of action. The EAB1 strain displayed a heightened ability to cross the epithelial barrier of human lung tissue, evade the immune system, and spread to lung tissues, ultimately resulting in cellular mortality. In contrast, the EAB2 strain strongly attached to epithelial cells, leading to increased synthesis of proinflammatory cytokines and chemokines. The genomic alterations and increased virulence observed in evolved strains during short-term evolution underscore the need for caution when handling these pathogens, as these risks persist even without antibiotic exposure.

Evaluate the Role of A. Baumannii in Promoting Inflammation and Meningitis-Related Genes in Various Immune Cells

The role of Acinetobacter baumannii in promoting inflammation in spleenocyte and lymph node cells has been studied by detecting the gene expression of four different genes that are related to meningitis, which was performed after treatment of laboratory animal cells with dead cells of A. baumannii, the results of gene expression by using Real Time- Quantitative PCR and graph prism software exhibited the presence of statistical differences between A. baumannii treated and non-treated laboratory animal cells. All genes showed an expression between one and a half (1.5) to more than 40 (>40) fold change. The NLRP has expression in the spleen and lymph nodes between the (2-10) folds, which increase after being activated with killed A. baumannii cells. NF Kappa B has expression in the spleen and lymph nodes between (1.5-6) fold increase after being activated with killed A. baumanniicells. GMCSF has expression in the spleen and lymph nodes between (30-40) fold increase after being activated with killed A. baumannii cells. IL-1B has expression in the spleen and lymph nodes between (4.5-40) fold increase after being activated with killed A. baumannii cells.

Adherence and cytotoxicity of Acinetobacter baumannii on human cervical carcinoma epithelial cells: Exploring the role of anti-OmpA antibodies

We investigated the invasion of Acinetobacter baumannii strains to epithelial cells and elucidated the role of antibodies against outer membrane protein A (OmpA). A. baumannii ATCC 19606 and clinical isolate 58ST were utilized. OmpA was expressed, purified, and administered to BALB/c mice, inducing anti-OmpA antibodies. OmpA cytotoxicity was evaluated. Two A. baumannii strains were selected to infect human cervical HeLa cells. Serum resistance was determined at sera dilutions. Adhesion, internalization, and proliferation of live and killed A. baumannii in HeLa cells were examined with and without anti-OmpA sera. HeLa cell viability was assessed with and without exposure of live A. baumannii strains to anti-OmpA sera. Cytoskeleton inhibitor experiments were conducted on epithelial cells to probe microfilament and microtubule involvement in A. baumannii invasion. OmpA prompted antibody production without toxicity in mice. A. baumannii strains displayed varying cell invasion abilities, notably the clinical strain exhibiting the highest invasion. A. baumannii cells localized within vacuoles during internalization, migrating towards the nucleus, using a zipper-like invasion process. Bacterial proliferation within host cells led to HeLa cell death. Pre-treatment with anti-OmpA antibodies significantly curbed adhesion and invasion of A. baumannii in HeLa cells. Microscopic imaging provided proof of the intracellular presence of A. baumannii in HeLa cells. In conclusion, the OmpA plays a crucial part in A. baumannii - epithelial cell interactions. The results add to our knowledge of pathogenesis during the initial stages of infection by A. baumannii.

Essential oils cause membrane disruption and autoaggregation of MDR Acinetobacter baumannii cells

Essential oils are promising antimicrobial agents against various bacteria. The aim of the study was to examine anti-Acinetobacter baumannii activity of nine essential oils and to elucidate essential oil mechanisms of action against this. In total, 8 out of 9 examined different essential oils exhibited activity against multidrug-resistant (MDR) A. baumannii isolates. The best anti-A. baumannii activity expressed Thymus serpyllum, Satureja hortensis and Oreganum majorana essential oil (MBC 1.29 to 2.58 µg mL−1), while essential oils of Menta x piperita, Hyssopus officinalis, Foeniculum vulgare and Artemisia dracunculus expressed the same effect in higher concentrations (1.80 to 3.80 µg mL−1). The effect of Juniperus sabina, Juniperus sibirica and S. hortensis essential oils against A. baumannii is for the first time reported here. The mechanisms of Myrtus communis, Eucalyptus camaldulensis, S. hortensis and T. serpyllum essential oils were further examined. The essential oil treatments led to concentration dependent leakage of biomolecules form the cells, causing an increase in the concentrations of lipids, carbohydrates, and proteins in the suspensions. Microstructural observations confirmed the essential oil effect on cell membranes and disruption the membrane integrity, with obvious leakage of intercellular substance, collapsed cells with perforations, debris presence and autoaggregated cells. The subinhibitory concentrations of oils did not obviously changed protein patterns determined by SDS-PAGE. These results indicate that the essential oils, particularly T. serpyllum, S. hortensis, M. communis and E. camaldulensis express its anti-A. baumannii activity via membrane disruption and increased membrane permeability, representing very promising alternative antibacterial agents against MDR wound isolates.

Direct analysis by ultra-high-resolution mass spectrometry of lipid A and phospholipids from Acinetobacter baumannii cells

Acinetobacter baumannii, classified as priority number one by the World Health Organization (WHO), is an opportunistic pathogen responsible for infection and is able to develop antibiotic resistance easily. Membranes are bacteria's first line of defense against external aggression, such as antibiotics. A chemical modification of a lipid family or a change in lipid composition can lead to resistance to antibiotics. In this work, we analyzed different A. baumannii strains from various environments with different antibiotic resistance profiles, using matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT-ICR MS). This study shows that it is possible to describe the main lipidome (phospholipids and lipid A) from the simple preparation of lysed cells, and that despite the complexity of the mixture. This ultra-high resolution mass spectrometry technique enables the separation of isobaric ion, to report a new class of lipids. Given its performance, this technique can be used to quickly and reliably characterize the lipidome of clinical strains from different environments.

Innate immune mechanisms promote human response to Acinetobacter baumannii infection.

Acinetobacter baumannii is an opportunistic Gram-negative bacterium representing one of the leading causes of ventilator-associated pneumonia. The development of pneumonia results from a complex interplay between pathogens and pulmonary innate mucosal immunity. Therefore, the knowledge of the host immune responses is pivotal for the development of effective therapeutics to treat A. baumannii infections. Previous studies were conducted using cell lines and animal models, but a comprehensive understanding of the interaction between A. baumannii and primary human immune cells is still lacking. To bridge this gap, we investigated the response of primary monocytes, macrophages, and dendritic cells to the A. baumannii-type strain and an epidemic clinical isolate. We found that all immune cells trigger different responses when interacting with A. baumannii. In particular, macrophages and monocytes mediate bacterial clearance, whereas monocytes and dendritic cells activate a late response through the production of cytokines, chemokines, and the expression of co-stimulatory molecules. The epidemic strain induces lower expression of interleukin-10 and CD80 compared with the type strain, potentially constituting two immune evasion strategies.

Evidence of Correlation between Membrane Phase Transition and Clonogenicity in Dehydrating Acinetobacter baumannii: A Combined Micro-Raman and AFM Study.

The Gram-negative bacterium Acinetobacter baumannii is one of the most resilient multidrug-resistant pathogens in hospitals. Among Gram-negative bacteria, it is particularly resistant to dehydration (anhydrobiosis), and this feature allows A. baumannii to persist in hospital environments for long periods, subjected to unfavorable conditions. We leverage the combination of μ-Raman spectroscopy and atomic force microscopy (AFM) to investigate the anhydrobiotic mechanisms in A. baumannii cells by monitoring the membrane (both inner and outer membranes) properties of four A. baumannii strains during a 16-week dehydration period and in response to temperature excursions. We noted that the membranes of A. baumannii remained intact during the dehydration period despite undergoing a liquid-crystal-to-gel-phase transition, accompanied by changes in the mechanical properties of the membrane. This was evident from the AFM images, which showed the morphology of the bacterial cells alongside modifications of their superficial mechanical properties, and from the alteration in the intensity ratio of μ-Raman features linked to the CH3 and CH2 symmetric stretching modes. Furthermore, employing a universal power law revealed a significant correlation between this ratio and bacterial fitness across all tested strains. Additionally, we subjected dry A. baumannii to a temperature-dependent experiment, the results of which supported the correlation between the Raman ratio and culturability, demonstrating that the phase transition becomes irreversible when A. baumannii cells undergo different temperature cycles. Besides the relevance to the present study, we argue that μ-Raman can be used as a powerful nondestructive tool to assess the health status of bacterial cells based on membrane properties with a relatively high throughput.

Targeting BamA, the essential component of the Acinetobacter baumannii β-barrel assembly machinery, hinders its ability to adhere to and invade human alveolar basal epithelial cell line

The lungs are commonly targeted by Acinetobacter baumannii. The human alveolar basal epithelial cell line, A549, serves as a valuable in vitro model for probing pathogen-cell dynamics. This study examined two Acinetobacter strains, ATCC 19606 and the clinical isolate 58ST, investigating their adherence, internalization, and cytotoxicity within the A549 cell line to illuminate pathogenic mechanisms. Anti-BamA antibodies were expressed, purified, and detected via indirect ELISA. The toxicity of BamA was assessed across BALB/c mice. Both A. baumannii strains were used to infect A549 cells to scrutinize cell invasion diversity. Serum resistance, biofilm creation and inhibition, adhesion, internalization, and intracellular proliferation of live and inactivated A. baumannii were probed with and without anti-BamA sera. A549 cell viability was evaluated in the presence of live A. baumannii and anti-BamA sera-exposed bacteria. Cytoskeleton inhibitor tests were conducted on epithelial cells. A. baumannii strains displayed differing cell invasion aptitudes, with the clinical variant manifesting the highest invasion capability. During internalization, A. baumannii cells localized within vacuoles and migrated towards the nucleus using a zipper-like invasion mechanism. Bacterial division inside host cells culminated in cell demise. Pre-treatment with anti-BamA antibodies substantially impeded A. baumannii's adherence and invasion in epithelial cells. Microscopic imaging validated the intracellular presence of A. baumannii in A549 cells, verifying their invasive potential and residency. These findings substantiate A. baumannii's capacity to proliferate in epithelial cells, with BamA pivotal role against A. baumannii-epithelial cell interplay. This study augments our insight into A. baumannii pathogenesis, facilitating the development of efficacious strategies against A. baumannii infections.

Investigating the Effectiveness of Ceragenins against Acinetobacter baumannii to Develop New Antimicrobial and Anti-Adhesive Strategies.

A growing body of experimental data indicates that ceragenins (CSAs), which mimic the physicochemical properties of the host's cationic antimicrobial peptide, hold promise for the development of a new group of broad-spectrum antimicrobials. Here, using a set of in vivo experiments, we assessed the potential of ceragenins in the eradication of an important etiological agent of nosocomial infections, Acinetobacter baumannii. Assessment of the bactericidal effect of ceragenins CSA-13, CSA-44, and CSA-131 on clinical isolates of A. baumannii (n = 65) and their effectiveness against bacterial cells embedded in the biofilm matrix after biofilm growth on abiotic surfaces showed a strong bactericidal effect of the tested molecules regardless of bacterial growth pattern. AFM assessment of bacterial cell topography, bacterial cell stiffness, and adhesion showed significant membrane breakdown and rheological changes, indicating the ability of ceragenins to target surface structures of A. baumannii cells. In the cell culture of A549 lung epithelial cells, ceragenin CSA-13 had the ability to inhibit bacterial adhesion to host cells, suggesting that it interferes with the mechanism of bacterial cell invasion. These findings highlight the potential of ceragenins as therapeutic agents in the development of antimicrobial strategies against bacterial infections caused by A. baumannii.

Antibacterial mechanism of Pseudomonas aeruginosa UKMP14T rhamnolipids against multidrug resistant Acinetobacter baumannii

Rhamnolipids, a major category of glycolipid biosurfactant, have recently gained enormous attention in medical field because of their relevance as effective antibacterial agents against a wide variety of pathogenic bacteria. Our previous studies have shown that rhamnolipids from an environmental isolate of Pseudomonas aeruginosa UKMP14T possess antibacterial, anti-adhesive and anti-biofilm activity against multidrug-resistant ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter sp.) pathogens. However, the mechanism of their antibacterial action remains unclear. Thus, this study aimed to elucidate the mechanism of the antibacterial action of P. aeruginosa UKMP14T rhamnolipids by studying the changes in cells of one of the ESKAPE pathogens, Acinetobacter baumannii, which is the most difficult strain to kill. Results revealed that rhamnolipid treatment rendered A. baumannii cells more hydrophobic as evaluated through contact angle measurements. It also induced the release of cellular proteins measuring 510 μg/mL at a rhamnolipid concentration of 1000 μg/mL. In addition, rhamnolipids were found to be bactericidal in their action as they could permeate the inner membranes, leading to a leak-out of nucleotides. More than 50 % of the cells were found to be killed upon 1000 μg/mL rhamnolipid treatment as observed through fluorescence microscopy. Other cellular changes such as irregular shape and size, membrane perturbations, clumping, shrinkage and physical damage were clearly visible in SEM, FESEM and laser micrographs. Furthermore, rhamnolipid treatment inhibited the levels of acyl-homoserine lactones (AHLs) in A. baumannii, which are vital for their biofilm formation and virulence. The obtained results indicate that P. aeruginosa UKMP14T rhamnolipids target outer and inner bacterial membranes through permeation, including physical damage to the cells, leading to cell leakage. Furthermore, AHL inhibition appears to be the mechanism behind their anti-biofilm action. All these observations can be correlated to rhamnolipids’ antibacterial effect against A. baumannii.

In vitro investigation into the dynamics between Acinetobacter baumannii, bacteriophage, and mammalian bronchial epithelial cells

Antibiotic resistance is escalating due to the rapid emergence of multidrug-resistant bacteria and the stagnation in new antibiotic development. Bacteriophage, a natural enemy of bacteria, has re-emerged as a promising alternative in the post-antibiotic era. However, none of the recently completed randomized, placebo-controlled, and double-blinded clinical trials on phage therapy could confirm its efficacy. One of the major impediments is the lack of understanding of the phage, bacteria, and host body interactions. Our work investigates the dynamics between Acinetobacter baumannii, bacteriophage, and a mammalian bronchial epithelial cell line (BEAS-2B) in a coculture system. Our results demonstrated that the bactericidal effect of bacteriophage could be augmented in the presence of non-mucus-producing epithelial cells (by 3 – 5 log). Adsorption study indicated that both phages and bacteria could adhere to the epithelial cells, subsequently promoting their contact and the phage lytic effect. The presence of epithelial cells could also effectively inhibit/delay the emergence of phage resistance. These findings suggested that evaluating the in vitro antibacterial efficiency of bacteriophage in the presence of mammalian cells may yield better predictions of the therapeutic outcomes of bacteriophage therapy.

In vitro and in silico assessment of anti-biofilm and anti-quorum sensing properties of 2,4-Di-tert butylphenol against Acinetobacter baumannii.

Introduction. Acinetobacter baumannii is a nosocomial pathogen with a high potential to cause food-borne infections. It is designated as a critical pathogen by the World Health Organization due to its multi-drug resistance and mortalities reported. Biofilm governs major virulence factors, which promotes drug resistance in A. baumannii. Thus, a compound with minimum selection pressure on the pathogen can be helpful to breach biofilm-related virulence.Hypothesis/Gap Statement. To identify anti-biofilm and anti-virulent metabolites from extracts of wild Mangifera indica (mango) brine pickle bacteria that diminishes pathogenesis and resistance of A. baumannii.Aim. This study reports anti-biofilm and anti-quorum sensing (QS) efficacy of secondary metabolites from bacterial isolates of fermented food origin.Method. Cell-free supernatants (CFS) of 13 bacterial isolates from fermented mango brine pickles were screened for their efficiency in inhibiting biofilm formation and GC-MS was used to identify its metabolites. Anti-biofilm metabolite was tested on early and mature biofilms, pellicle formation, extra polymeric substances (EPS), cellular adherence, motility and resistance of A. baumannii. Gene expression and in silico studies were also carried out to validate the compounds efficacy.Results. CFS of TMP6b identified as Bacillus vallismortis, inhibited biofilm production (83.02 %). Of these, major compound was identified as 2,4-Di-tert-butyl phenol (2,4-DBP). At sub-lethal concentrations, 2,4-DBP disrupted both early and mature biofilm formation. Treatment with 2,4-DBP destructed in situ biofilm formed on glass and plastic. In addition, key virulence traits like pellicle (77.5 %), surfactant (95.3 %), EPS production (3-fold) and cell adherence (65.55 %) reduced significantly. A. baumannii cells treated with 2,4-DBP showed enhanced sensitivity towards antibiotics, oxide radicals and blood cells. Expression of biofilm-concomitant virulence genes like csuA/B, pgaC, pgaA, bap, bfmR, katE and ompA along with QS genes abaI, abaR significantly decreased. The in silico studies further validated the higher binding affinity of 2,4-DBP to the AbaR protein than the cognate ligand molecule.Conclusion. To our knowledge, this is the first report to demonstrate 2,4- DBP has anti-pathogenic potential alone and with antibiotics by in vitro, and in silico studies against A. baumannii. It also indicates its potential use in therapeutics and bio-preservatives.

Simultaneous Bdellovibrio bacteriovorus–Bacteriophage dosing with SODIS for treatment of environmental water sources

The predatory bacterium Bdellovibrio bacteriovorus HW1 and bacteriophage ABTW1 were applied simultaneously, with solar disinfection (SODIS), for the reduction of Acinetobacter baumannii in environmental water sources. The B. bacteriovorus HW1 and ABTW1 dosing protocol was optimised, with results indicating that the simultaneous application of B. bacteriovorus HW1 and ABTW1 at 0 h resulted in the greatest log reduction (5.66 and 3.97 log based on CFU/mL and GC/mL, respectively) in A. baumannii over the 96 h co-culture period. Similar log reductions (5.24 and 3.85 log reduction based on CFU/mL and GC/mL, respectively) were however, obtained for the initial dosing of B. bacteriovorus at 0 h, and addition of bacteriophage ABTW1 at 24 h. A lyophilised bacteriophage formulation (0.5 M sucrose and 1% gelatine) was then compared to the standard liquid bacteriophage inoculum, with B. bacteriovorus, in upscaled volume trials. However, as similar A. baumannii log reduction results were obtained, the standard bacteriophage liquid inoculum, with B. bacteriovorus, was assessed as a pre-treatment to 6 h SODIS. The B. bacteriovorus HW1-bacteriophage ABTW1 combination as well as the B. bacteriovorus HW1 only pre-treatment, both with SODIS, significantly reduced the A. baumannii cell counts (8.32 and 8.38 log, respectively) to below detection limit (BDL). Molecular analysis however, showed that A. baumannii persisted (∼ 3 log reduction) after the application of all treatment protocols. It is thus recommended that the efficiency of the B. bacteriovorus-bacteriophage combination is further optimised to improve the lytic effect of both biological control agents, particularly the bacteriophage, on their prey.

Genetic synergy between Acinetobacter baumannii undecaprenyl phosphate biosynthesis and the Mla system impacts cell envelope and antimicrobial resistance.

Acinetobacter baumannii is a Gram-negative bacterial pathogen that poses a major health concern due to increasing multidrug resistance. The Gram-negative cell envelope is a key barrier to antimicrobial entry and includes an inner and outer membrane. The maintenance of lipid asymmetry (Mla) system is the main homeostatic mechanism by which Gram-negative bacteria maintain outer membrane asymmetry. Loss of the Mla system in A. baumannii results in attenuated virulence and increased susceptibility to membrane stressors and some antibiotics. We recently reported two strain variants of the A. baumannii type strain ATCC 17978: 17978VU and 17978UN. Here, ∆mlaF mutants in the two ATCC 17978 strains display different phenotypes for membrane stress resistance, antibiotic resistance, and pathogenicity in a murine pneumonia model. Although allele differences in obgE were previously reported to synergize with ∆mlaF to affect growth and stringent response, obgE alleles do not affect membrane stress resistance. Instead, a single-nucleotide polymorphism (SNP) in the essential gene encoding undecaprenyl pyrophosphate (Und-PP) synthase, uppS, results in decreased enzymatic rate and decrease in total Und-P levels in 17978UN compared to 17978VU. The UppSUN variant synergizes with ∆mlaF to reduce capsule and lipooligosaccharide (LOS) levels, increase susceptibility to membrane stress and antibiotics, and reduce persistence in a mouse lung infection. Und-P is a lipid glycan carrier required for the biosynthesis of A. baumannii capsule, cell wall, and glycoproteins. These findings uncover synergy between Und-P and the Mla system in maintaining the A. baumannii cell envelope and antibiotic resistance.IMPORTANCEAcinetobacter baumannii is a critical threat to global public health due to its multidrug resistance and persistence in hospital settings. Therefore, novel therapeutic approaches are urgently needed. We report that a defective undecaprenyl pyrophosphate synthase (UppS) paired with a perturbed Mla system leads to synthetically sick cells that are more susceptible to clinically relevant antibiotics and show reduced virulence in a lung infection model. These results suggest that targeting UppS or undecaprenyl species and the Mla system may resensitize A. baumannii to antibiotics in combination therapies. This work uncovers a previously unknown synergistic relationship in cellular envelope homeostasis that could be leveraged for use in combination therapy against A. baumannii.

The metabolite vanillic acid regulates Acinetobacter baumannii surface attachment.

The nosocomial bacterium Acinetobacter baumannii is protected from antibiotic treatment by acquiring antibiotic resistances and by forming biofilms. Cell attachment, one of the first steps in biofilm formation, is normally induced by environmental metabolites. We hypothesized that vanillic acid (VA), the oxidized form of vanillin and a widely available metabolite, may play a role in A. baumannii cell attachment. We first discovered that A. baumannii actively breaks down VA through the evolutionarily conserved vanABKP genes. These genes are under the control of the repressor VanR, which we show binds directly to VanR binding sites within the vanABKP genes bidirectional promoter. VA in turn counteracts VanR inhibition. We identified a VanR binding site and searched for it throughout the genome, especially in pili encoding promoter genes. We found a VanR binding site in the pilus encoding csu operon promoter and showed that VanR binds specifically to it. As expected, a strain lacking VanR overproduces Csu pili and makes robust biofilms. Our study uncovers the role that VA plays in facilitating the attachment of A. baumannii cells to surfaces, a crucial step in biofilm formation. These findings provide valuable insights into a previously obscure catabolic pathway with significant clinical implications.

Acinetobacter baumannii Survival under Infection-Associated Stresses Depends on the Expression of Resistance-Nodulation-Division and Major Facilitator Superfamily Efflux Pumps.

Multidrug efflux transporters are major contributors to the antibiotic resistance of Acinetobacter baumannii in clinical settings. Previous studies showed that these transporters are tightly integrated into the physiology of A. baumannii and have diverse functions. However, for many of the efflux pumps, such functions remain poorly defined. In this study, we characterized two putative drug efflux pumps, AmfAB and AmfCD (Acinetobacter Major Facilitator), that are homologous to EmrAB-like transporters from Escherichia coli and other Gram-negative bacteria. These pumps comprise the Major Facilitator Superfamily (MFS) transporters AmfB and AmfD and the periplasmic membrane fusion proteins AmfA and AmfC, respectively. We inactivated and overproduced these pumps in the wild-type ATCC 17978 strain and its derivative strains lacking the major efflux pumps from the Resistance-Nodulation-Division (RND) superfamily and characterized antibiotic susceptibilities and growth of the strains under stresses typical during human infections. We found that neither AmfAB nor AmfCD contribute to the antibiotic non-susceptibility phenotypes of A. baumannii. The two pumps, however, are critical for the adaptation and growth of the bacterium under acidic stress, whereas AmfCD also contributes to growth under conditions of low iron, high temperature, and in the presence of bile salts. These functions are dependent on the presence of the RND pumps, the inactivation of which further diminishes A. baumannii survival and growth. Our results suggest that MFS transporters contribute to stress survival by affecting the permeability properties of the A. baumannii cell envelope.

Proguanil and chlorhexidine augment the antibacterial activities of clarithromycin and rifampicin against Acinetobacter baumannii

The emergence of Acinetobacter baumannii infections as a significant healthcare concern in hospital settings, coupled with their association with poorer clinical outcomes, has prompted extensive investigation into novel therapeutic agents and innovative treatment strategies. Proguanil and chlorhexidine, both categorized as biguanide compounds, have displayed clinical efficacy as antimalarial and topical antibacterial agents, respectively. In this study, we conducted an investigation to assess the effectiveness of combining proguanil and chlorhexidine with clarithromycin or rifampicin against both laboratory strains and clinical isolates of A. baumannii. The combination therapy demonstrated rapid bactericidal activity against planktonic multidrug-resistant A. baumannii, exhibiting efficacy in eradicating mature biofilms and impeding the development of antibiotic resistance in vitro. Additionally, when administered in conjunction with clarithromycin or rifampicin, proguanil enhanced the survival rate of mice afflicted with intraperitoneal A. baumannii infections, and chlorhexidine expedited wound healing in mice with skin infections. These findings are likely attributable to the disruption of A. baumannii cell membrane integrity by proguanil and chlorhexidine, resulting in heightened membrane permeability and enhanced intracellular accumulation of clarithromycin and rifampicin. Overall, this study underscores the potential of employing proguanil and chlorhexidine in combination with specific antibiotics to effectively combat A. baumannii infections and improve treatment outcomes in clinically challenging scenarios.