scholarly journals Defining Gene-Phenotype Relationships in Acinetobacter baumannii through One-Step Chromosomal Gene Inactivation

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Ashley T. Tucker ◽  
Emily M. Nowicki ◽  
Joseph M. Boll ◽  
Gregory A. Knauf ◽  
Nora C. Burdis ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Antibiotic Resistance ◽  
Acinetobacter Baumannii ◽  
Genome Editing ◽  
Biofilm Formation ◽  
Content Type ◽  
Stress Protection ◽  
Biocide Resistance ◽  
Hospital Acquired ◽  
Oxidative Stress Protection

ABSTRACTRates of infection with hospital-acquiredAcinetobacter baumanniihave exploded over the past decade due to our inability to limit persistence and effectively treat disease.A. baumanniiquickly acquires antibiotic resistance, and its genome encodes mechanisms to tolerate biocides and desiccation, which enhance its persistence in hospital settings. With depleted antibiotic options, new methods to treatA. baumanniiinfections are desperately needed. A comprehensive understanding detailingA. baumanniicellular factors that contribute to its resiliency at genetic and mechanistic levels is vital to the development of new treatment options. Tools to rapidly dissect theA. baumanniigenome will facilitate this goal by quickly advancing our understanding ofA. baumanniigene-phenotype relationships. We describe here a recombination-mediated genetic engineering (recombineering) system for targeted genome editing ofA. baumannii. We have demonstrated that this system can perform directed mutagenesis on wide-ranging genes and operons and is functional in various strains ofA. baumannii, indicating its broad application. We utilized this system to investigate key gene-phenotype relationships inA. baumanniibiology important to infection and persistence in hospitals, including oxidative stress protection, biocide resistance mechanisms, and biofilm formation. In addition, we have demonstrated that both the formation and movement of type IV pili play an important role inA. baumanniibiofilm.IMPORTANCEAcinetobacter baumanniiis the causative agent of hospital-acquired infections, including pneumonia and serious blood and wound infections.A. baumanniiis an emerging pathogen and has become a threat to public health because it quickly develops antibiotic resistance, making treatment difficult or impossible. While the threat ofA. baumanniiis well recognized, our understanding of even its most basic biology lags behind. Analysis ofA. baumanniicellular functions to identify potential targets for drug development has stalled due in part to laborious genetic techniques. Here we have pioneered a novel recombineering system that facilitates efficient genome editing inA. baumanniiby single PCR products. This technology allows for rapid genome editing to quickly ascertain gene-phenotype relationships. To demonstrate the power of recombineering in dissectingA. baumanniibiology, we use this system to establish key gene-phenotype relationships important to infection and persistence in hospitals, including oxidative stress protection, biocide resistance, and biofilm formation.

scholarly journals Lon Protease Has Multifaceted Biological Functions inAcinetobacter baumannii

2018 ◽  
Vol 201 (2) ◽  
Author(s):  
Carly Ching ◽  
Brendan Yang ◽  
Chineme Onwubueke ◽  
David Lazinski ◽  
Andrew Camilli ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Biofilm Formation ◽  
Developmental Process ◽  
Cell Envelope ◽  
Opportunistic Pathogen ◽  
Lon Protease ◽  
Content Type ◽  
Hospital Acquired Infections ◽  
Biofilm Development ◽  
Hospital Acquired

ABSTRACTAcinetobacter baumanniiis a Gram-negative opportunistic pathogen that is known to survive harsh environmental conditions and is a leading cause of hospital-acquired infections. Specifically, multicellular communities (known as biofilms) ofA. baumanniican withstand desiccation and survive on hospital surfaces and equipment. Biofilms are bacteria embedded in a self-produced extracellular matrix composed of proteins, sugars, and/or DNA. Bacteria in a biofilm are protected from environmental stresses, including antibiotics, which provides the bacteria with selective advantage for survival. Although some gene products are known to play roles in this developmental process inA. baumannii, mechanisms and signaling remain mostly unknown. Here, we find that Lon protease inA. baumanniiaffects biofilm development and has other important physiological roles, including motility and the cell envelope. Lon proteases are found in all domains of life, participating in regulatory processes and maintaining cellular homeostasis. These data reveal the importance of Lon protease in influencing keyA. baumanniiprocesses to survive stress and to maintain viability.IMPORTANCEAcinetobacter baumanniiis an opportunistic pathogen and is a leading cause of hospital-acquired infections.A. baumanniiis difficult to eradicate and to manage, because this bacterium is known to robustly survive desiccation and to quickly gain antibiotic resistance. We sought to investigate biofilm formation inA. baumannii, since much remains unknown about biofilm formation in this bacterium. Biofilms, which are multicellular communities of bacteria, are surface attached and difficult to eliminate from hospital equipment and implanted devices. Our research identifies multifaceted physiological roles for the conserved bacterial protease Lon inA. baumannii. These roles include biofilm formation, motility, and viability. This work broadly affects and expands understanding of the biology ofA. baumannii, which will permit us to find effective ways to eliminate the bacterium.

scholarly journals Proteomic and Systematic Functional Profiling Unveils Citral Targeting Antibiotic Resistance, Antioxidant Defense, and Biofilm-Associated Two-Component Systems of Acinetobacter baumannii To Encumber Biofilm and Virulence Traits

mSystems ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Anthonymuthu Selvaraj ◽  
Alaguvel Valliammai ◽  
Pandiyan Muthuramalingam ◽  
Sivasamy Sethupathy ◽  
Ganapathy Ashwinkumar Subramenium ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Acinetobacter Baumannii ◽  
Biofilm Formation ◽  
Antioxidant Defense ◽  
Time Of Flight ◽  
Differentially Expressed ◽  
Secretion Systems ◽  
Content Type ◽  
Network Analyses ◽  
Wide Range

ABSTRACT Acinetobacter baumannii has been reported as a multidrug-resistant bacterium due to biofilms and antimicrobial resistance mechanisms. Hence, novel therapeutic strategies are necessary to overcome A. baumannii infections. This study revealed that citral at 200 μg/ml attenuated A. baumannii biofilms by up to 90% without affecting viability. Furthermore, microscopic analyses and in vitro assays confirmed the antibiofilm efficacy of citral. The global effect of citral on A. baumannii was evaluated by proteomic, transcriptional, and in silico approaches. Two-dimensional (2D) gel electrophoresis and matrix-assisted laser desorption ionization–time of flight/time of flight (MALDI-TOF/TOF) analyses were used to assess the effect of citral on the A. baumannii cellular proteome. Quantitative real-time PCR (qPCR) analysis was done to validate the proteomic data and identify the differentially expressed A. baumannii genes. Protein-protein interactions, gene enrichment, and comparative gene network analyses were performed to explore the interactions and functional attributes of differentially expressed proteins of A. baumannii. Global omics-based analyses revealed that citral targeted various mechanisms such as biofilm formation, antibiotic resistance, antioxidant defense, iron acquisition, and type II and type IV secretion systems. The results of antioxidant analyses and antibiotic sensitivity, blood survival, lipase, and hemolysis assays validated the proteomic results. Cytotoxicity analysis showed a nontoxic effect of citral on peripheral blood mononuclear cells (PBMCs). Overall, the current study unveiled that citral has multitarget efficacy to inhibit the biofilm formation and virulence of A. baumannii. IMPORTANCE Acinetobacter baumannii is a nosocomial-infection-causing bacterium and also possesses multidrug resistance to a wide range of conventional antibiotics. The biofilm-forming ability of A. baumannii plays a major role in its resistance and persistence. There is an alarming need for novel treatment strategies to control A. baumannii biofilm-associated issues. The present study demonstrated the strong antibiofilm and antivirulence efficacy of citral against A. baumannii. In addition, proteomic analysis revealed the multitarget potential of citral against A. baumannii. Furthermore, citral treatment enhances the susceptibility of A. baumannii to the host innate immune system and reactive oxygen species (ROS). Cytotoxicity analysis revealed the nonfatal effect of citral on human PBMCs. Therefore, citral could be the safest therapeutic compound and can be taken for further clinical evaluation for the treatment of biofilm-associated infections by A. baumannii.

scholarly journals Phase-Variable Control of Multiple Phenotypes in Acinetobacter baumannii Strain AB5075

2015 ◽  
Vol 197 (15) ◽  
pp. 2593-2599 ◽  
Author(s):  
Kyle A. Tipton ◽  
Daniela Dimitrova ◽  
Philip N. Rather
Keyword(s):  
Antibiotic Resistance ◽  
Acinetobacter Baumannii ◽  
Cell Density ◽  
Biofilm Formation ◽  
Galleria Mellonella ◽  
Phase Variation ◽  
Phase Variable ◽  
Dependent Manner ◽  
Content Type ◽  
Variable Control

ABSTRACTAcinetobacter baumanniistrain AB5075 produces colonies with two opacity phenotypes, designated opaque and translucent. These phenotypes were unstable and opaque and translucent colony variants were observed to interconvert at high frequency, suggesting that a phase-variable mechanism was responsible. The frequency of phase variation both within colonies and in broth cultures increased in a cell density-dependent manner and was mediated by the accumulation of an extracellular factor. This factor was distinct from the knownA. baumanniisignaling molecule 3-OH C12-homoserine lactone. Opaque and translucent colony variants exhibited a number of phenotypic differences, including cell morphology, surface motility, biofilm formation, antibiotic resistance, and virulence in aGalleria mellonellamodel. Additional clinical isolates exhibited a similar phase-variable control of colony opacity, suggesting that this may be a common feature ofA. baumannii.IMPORTANCEA novel phase-variable mechanism has been identified inAcinetobacter baumanniithat results in an interconversion between opaque and translucent colony phenotypes. This phase variation also coordinately regulates motility, cell shape, biofilm formation, antibiotic resistance, and virulence. The frequency of phase variation is increased at high cell density via a diffusible extracellular signal. To our knowledge, this report presents the first example of phase variation inA. baumanniiand also the first example of quorum sensing-mediated control of phase variation in a bacterium. The findings are important, as this phase-variable mechanism can be identified only via changes in colony opacity using oblique light; therefore, many researchers studyingA. baumanniimay unknowingly be working with different colony variants.

scholarly journals Contribution of Resistance-Nodulation-Cell Division Efflux Systems to Antibiotic Resistance and Biofilm Formation in Acinetobacter baumannii

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Eun-Jeong Yoon ◽  
Yassine Nait Chabane ◽  
Sylvie Goussard ◽  
Erik Snesrud ◽  
Patrice Courvalin ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Cell Division ◽  
Acinetobacter Baumannii ◽  
Resistance Gene ◽  
Biofilm Formation ◽  
Hospital Environment ◽  
Intrinsic Resistance ◽  
Content Type ◽  
Clinical Resistance ◽  
Isogenic Mutants

ABSTRACTAcinetobacter baumanniiis a nosocomial pathogen of increasing importance due to its multiple resistance to antibiotics and ability to survive in the hospital environment linked to its capacity to form biofilms. To fully characterize the contribution of AdeABC, AdeFGH, and AdeIJK resistance-nodulation-cell division (RND)-type efflux systems to acquired and intrinsic resistance, we constructed, from an entirely sequenced susceptibleA. baumanniistrain, a set of isogenic mutants overexpressing each system following introduction of a point mutation in their cognate regulator or a deletion for the pump by allelic replacement. Pairwise comparison of every derivative with the parental strain indicated that AdeABC and AdeFGH are tightly regulated and contribute to acquisition of antibiotic resistance when overproduced. AdeABC had a broad substrate range, including β-lactams, fluoroquinolones, tetracyclines-tigecycline, macrolides-lincosamides, and chloramphenicol, and conferred clinical resistance to aminoglycosides. Importantly, when combined with enzymatic resistance to carbapenems and aminoglycosides, this pump contributed in a synergistic fashion to the level of resistance of the host. In contrast, AdeIJK was expressed constitutively and was responsible for intrinsic resistance to the same major drug classes as AdeABC as well as antifolates and fusidic acid. Surprisingly, overproduction of AdeABC and AdeIJK altered bacterial membrane composition, resulting in decreased biofilm formation but not motility. Natural transformation and plasmid transfer were diminished in recipients overproducing AdeABC. It thus appears that alteration in the expression of efflux systems leads to multiple changes in the relationship between the host and its environment, in addition to antibiotic resistance.IMPORTANCEIncreased expression of chromosomal genes for RND-type efflux systems plays a major role in bacterial multidrug resistance.Acinetobacter baumanniihas recently emerged as an important human pathogen responsible for epidemics of hospital-acquired infections. Besides its remarkable ability to horizontally acquire resistance determinants, it has a broad intrinsic resistance due to low membrane permeability, endogenous resistance genes, and antibiotic efflux. The study of isogenic mutants from a susceptibleA. baumanniiclinical isolate overproducing or deleted for each of the three major RND-type pumps demonstrated their major contribution to intrinsic resistance and to the synergism between overproduction of an efflux system and acquisition of a resistance gene. We have also shown that modulation of expression of the structural genes for the efflux systems results in numerous alterations in membrane-associated cellular functions, in particular, in a decrease in biofilm formation and resistance gene acquisition.

scholarly journals Detection of biofilm formation and correlation with antibiotic resistance in Acinetobacter baumannii

2020 ◽  
Vol 11 (SPL4) ◽  
pp. 2474-2480
Author(s):  
Anitha M ◽  
Jagatheeswary P A T ◽  
Sumathi G ◽  
Kamini B
Keyword(s):  
Antibiotic Resistance ◽  
Acinetobacter Baumannii ◽  
Biofilm Formation ◽  
Screening Method ◽  
Strong Relationship ◽  
Microtiter Plate ◽  
Diffusion Method ◽  
Drug Resistant ◽  
Biofilm Production ◽  
Hospital Acquired

Acinetobacter baumannii is a major cause of hospital acquired infections worldwide and is associated with resistance to routinely used antibiotics. Many clinical isolates of Acinetobacter baumannii  are found to be biofilm producers. Hence there is difficulty in treating patients with Multi Drug Resistant Acinetobacter baumannii  (MDRAB). This present study aimed to study antibiotic resistance of A. baumannii isolates  and to evaluate the biofilm formation of Acinetobacter baumannii  by Tube Method (TM) and Microtiter Plate Method(MTPM).In this study, 73 A. baumannii  isolates of various clinical specimens were evaluated. Confirmation was done through conventional methods. Testing for antimicrobial susceptibility was done by Kirby Bauer disc diffusion method. Biofilm formation was studied by TM and MTPM. Of the 73 isolates, 26(36%) were from urine, 19 (26%) from pus, 17 (23%) from sputum and 11 (15%) from other miscellaneous(body fluids excluding blood), out of which 81%(59/73) isolates were Multi Drug Resistant (MDR). 63% and 84% of isolates showed biofilm production in TM and MTPM, respectively. When comparing these two methods, MTPM assay was better than TM. Presence of a strong relationship between biofilm formation and MDRAB has been confirmed by the present study. Both methods used for detection of biofilm formation were found to be statistically significant. Sensitivity of MTPM was more than TM, which is supported by higher Positive Predictive Value of 87.5%. Therefore MTPM is a better method than TM and can be used as a screening method to detect biofilm production.

scholarly journals Interaction of Acinetobacter baumannii with Human Serum Albumin: Does the Host Determines the Outcome?

Antibiotics ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 833
Author(s):  
Camila Pimentel ◽  
Casin Le ◽  
Marisel R. Tuttobene ◽  
Tomas Subils ◽  
Krisztina M. Papp-Wallace ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Human Serum Albumin ◽  
Quorum Sensing ◽  
Serum Albumin ◽  
Human Serum ◽  
Acinetobacter Baumannii ◽  
Biofilm Formation ◽  
Model Systems ◽  
Expression Levels ◽  
Multiple Drugs

Acinetobacter baumannii has become a serious threat to human health due to its extreme antibiotic resistance, environmental persistence, and capacity to survive within the host. Two A. baumannii strains, A118 and AB5075, commonly used as model systems, and three carbapenem-resistant strains, which are becoming ever more dangerous due to the multiple drugs they can resist, were exposed to 3.5% human serum albumin (HSA) and human serum (HS) to evaluate their response with respect to antimicrobial resistance, biofilm formation, and quorum sensing, all features responsible for increasing survival and persistence in the environment and human body. Expression levels of antibiotic resistance genes were modified differently when examined in different strains. The cmlA gene was upregulated or downregulated in conditions of exposure to 3.5% HSA or HS depending on the strain. Expression levels of pbp1 and pbp3 tended to be increased by the presence of HSA and HS, but the effect was not seen in all strains. A. baumannii A118 growing in the presence of HS did not experience increased expression of these genes. Aminoglycoside-modifying enzymes were also expressed at higher or lower levels in the presence of HSA or HS. Still, the response was not uniform; in some cases, expression was enhanced, and in other cases, it was tapered. While A. baumannii AB5075 became more susceptible to rifampicin in the presence of 3.5% HSA or HS, strain A118 did not show any changes. Expression of arr2, a gene involved in resistance to rifampicin present in A. baumannii AMA16, was expressed at higher levels when HS was present in the culture medium. HSA and HS reduced biofilm formation and production of N-Acyl Homoserine Lactone, a compound intimately associated with quorum sensing. In conclusion, HSA, the main component of HS, stimulates a variety of adaptative responses in infecting A. baumannii strains.

Caveolin-1 promotes radioresistance in rhabdomyosarcoma through increased oxidative stress protection and DNA repair

2021 ◽  
Vol 505 ◽  
pp. 1-12
Author(s):  
Silvia Codenotti ◽  
Francesco Marampon ◽  
Luca Triggiani ◽  
Marco Lorenzo Bonù ◽  
Stefano Maria Magrini ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Repair ◽  
Caveolin 1 ◽  
Stress Protection ◽  
Oxidative Stress Protection

scholarly journals Engineered EVs for Oxidative Stress Protection

2021 ◽  
Vol 14 (8) ◽  
pp. 703
Author(s):  
Anna Maria Tolomeo ◽  
Santina Quarta ◽  
Alessandra Biasiolo ◽  
Mariagrazia Ruvoletto ◽  
Michela Pozzobon ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Extracellular Vesicles ◽  
Transgene Expression ◽  
Hepatoma Cell ◽  
Serine Protease Inhibitor ◽  
Protein Product ◽  
Hepatoma Cell Line ◽  
Cytoprotective Activity ◽  
Stress Protection ◽  
Oxidative Stress Protection

Extracellular vesicles (EVs) are increasingly studied as vectors for drug delivery because they can transfer a variety of molecules across biological barriers. SerpinB3 is a serine protease inhibitor that has shown a protective anti-apoptotic function in a variety of stressful conditions. The aim of this study was to evaluate protection from oxidative stress-induced damage, using extracellular vesicles that overexpress SerpinB3 (EVs-SB3) in order to enhance the effect of extracellular vesicles on cellular homeostasis. EVs-SB3s were obtained from HepG2 cells engineered to overexpress SerpinB3 and they revealed significant proteomic changes, mostly characterized by a reduced expression of other proteins compared with EVs from non-engineered cells. These EV preparations showed a significantly higher protection from H2O2 induced oxidative stress in both the hepatoma cell line and in primary cardiomyocytes, compared to cells treated with naïve EVs or SerpinB3 alone, used at the same concentration. In conclusion, the induction of SerpinB3 transgene expression results in the secretion of EVs enriched with the protein product that exhibits enhanced cytoprotective activity, compared with naïve EVs or the nude SerpinB3 protein.

scholarly journals Growth Retardation, Reduced Invasiveness, and Impaired Colistin-Mediated Cell Death Associated with Colistin Resistance Development in Acinetobacter baumannii

2013 ◽  
Vol 58 (2) ◽  
pp. 828-832 ◽  
Author(s):  
Spyros Pournaras ◽  
Aggeliki Poulou ◽  
Konstantina Dafopoulou ◽  
Yassine Nait Chabane ◽  
Ioulia Kristo ◽  
...  
Keyword(s):  
Cell Death ◽  
Acinetobacter Baumannii ◽  
Biofilm Formation ◽  
Bloodstream Infections ◽  
Single Amino Acid ◽  
Colistin Resistance ◽  
Resistance Development ◽  
Content Type ◽  
Alkyl Hydroperoxide ◽  
Severe Soft Tissue

ABSTRACTTwo colistin-susceptible/colistin-resistant (Cols/Colr) pairs ofAcinetobacter baumanniistrains assigned to international clone 2, which is prevalent worldwide, were sequentially recovered from two patients after prolonged colistin administration. Compared with the respective Colsisolates (Ab248 and Ab299, both having a colistin MIC of 0.5 μg/ml), both Colrisolates (Ab249 and Ab347, with colistin MICs of 128 and 32 μg/ml, respectively) significantly overexpressedpmrCABgenes, had single-amino-acid shifts in the PmrB protein, and exhibited significantly slower growth. The Colrisolate Ab347, tested by proteomic analysis in comparison with its Colscounterpart Ab299, underexpressed the proteins CsuA/B and C from thecsuoperon (which is necessary for biofilm formation). This isolate also underexpressed aconitase B and different enzymes involved in the oxidative stress response (KatE catalase, superoxide dismutase, and alkyl hydroperoxide reductase), suggesting a reduced response to reactive oxygen species (ROS) and, consequently, impaired colistin-mediated cell death through hydroxyl radical production. Colsisolates that were indistinguishable by macrorestriction analysis from Ab299 caused six sequential bloodstream infections, and isolates indistinguishable from Ab248 caused severe soft tissue infection, while Colrisolates indistinguishable from Ab347 and Ab249 were mainly colonizers. In particular, a Colsisolate identical to Ab299 was still invading the bloodstream 90 days after the colonization of this patient by Colrisolates. These observations indicate considerably lower invasiveness ofA. baumanniiclinical isolates following the development of colistin resistance.

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