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Transcriptional analyses of Acinetobacter baumannii ATCC 17978 showed that the expression of A1S_2091 was enhanced in cells cultured in darkness at 24°C through a process that depended on the BlsA photoreceptor. Disruption of A1S_2091, a component of the A1S_2088-A1S_2091 polycistronic operon predicted to code for a type I chaperone/usher pilus assembly system, abolished surface motility and pellicle formation but significantly enhanced biofilm formation on plastic by bacteria cultured in darkness. Based on these observations, the A1S_2088-A1S_2091 operon was named the p hoto r egulated p ilus ABCD ( prpABCD ) operon, with A1S_2091 coding for the PrpA pilin subunit. Unexpectedly, comparative analyses of ATCC 17978 and prpA isogenic mutant cells cultured at 37°C showed the expression of light-regulated biofilm biogenesis and motility functions under a temperature condition that drastically affects BlsA production and its light-sensing activity. These assays also suggest that ATCC 17978 cells produce alternative light-regulated adhesins and/or pilus systems that enhance bacterial adhesion and biofilm formation at both 24°C and 37°C on plastic as well as on the surface of polarized A549 alveolar epithelial cells, where the formation of bacterial filaments and cell chains was significantly enhanced. The inactivation of prpA also resulted in a significant reduction in virulence when tested by using the Galleria mellonella virulence model. All these observations provide strong evidence showing the capacity of A. baumannii to sense light and interact with biotic and abiotic surfaces using undetermined alternative sensing and regulatory systems as well as alternative adherence and motility cellular functions that allow this pathogen to persist in different ecological niches., (Copyright © 2018 American Society for Microbiology.)

The rise of drug-resistant bacterial infections coupled with decreasing antibiotic efficacy poses a significant challenge to global health care. Acinetobacter baumannii is an insidious, emerging bacterial pathogen responsible for severe nosocomial infections aided by its ability to form biofilms. The response regulator BfmR, from the BfmR/S two-component system, is the master regulator of biofilm initiation in A. baumannii and is a tractable therapeutic target. Here we present the structure of A. baumannii BfmR using a hybrid approach combining X-ray crystallography, nuclear magnetic resonance spectroscopy, chemical crosslinking mass spectrometry, and molecular modeling. We also show that BfmR binds the previously proposed bfmRS promoter sequence with moderate affinity. While BfmR shares many traits with other OmpR/PhoB family response regulators, some unusual properties were observed. Most importantly, we observe that when phosphorylated, BfmR binds this promoter sequence with a lower affinity than when not phosphorylated. All other OmpR/PhoB family members studied to date show an increase in DNA-binding affinity upon phosphorylation. Understanding the structural and biochemical mechanisms of BfmR will aid in the development of new antimicrobial therapies., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Many strains of Acinetobacter baumannii have been described as being able to form biofilm. Small non-coding RNAs (sRNAs) control gene expression in many regulatory circuits in bacteria. The aim of the present work was to provide a global description of the sRNAs produced both by planktonic and biofilm-associated (sessile) cells of A. baumannii ATCC 17978, and to compare the corresponding gene expression profiles to identify sRNAs molecules associated to biofilm formation and virulence. sRNA was extracted from both planktonic and sessile cells and reverse transcribed. cDNA was subjected to 454-pyrosequencing using the GS-FLX Titanium chemistry. The global analysis of the small RNA transcriptome revealed different sRNA expression patterns in planktonic and biofilm associated cells, with some of the transcripts only expressed or repressed in sessile bacteria. A total of 255 sRNAs were detected, with 185 of them differentially expressed in the different types of cells. A total of 9 sRNAs were expressed only in biofilm cells, while the expression of other 21 coding regions were repressed only in biofilm cells. Strikingly, the expression level of the sRNA 13573 was 120 times higher in biofilms than in planktonic cells, an observation that prompted us to further investigate the biological role of this non-coding transcript. Analyses of an isogenic mutant and over-expressing strains revealed that the sRNA 13573 gene is involved in biofilm formation and attachment to A549 human alveolar epithelial cells. The present work serves as a basis for future studies examining the complex regulatory network that regulate biofilm biogenesis and attachment to eukaryotic cells in A. baumannii ATCC 17978.

Acinetobacter baumannii is a nosocomial pathogen that has a considerable ability to survive in the hospital environment partly due to its capacity to form biofilms. The first step in the process of establishing an infection is adherence of the bacteria to target cells. Chaperone-usher pili assembly systems are involved in pilus biogenesis pathways that play an important role in adhesion to host cells and tissues as well as medically relevant surfaces. After screening a collection of strains, a biofilm hyper-producing A. baumannii strain (MAR002) was selected to describe potential targets involved in pathogenicity. MAR002 showed a remarkable ability to form biofilm and attach to A549 human alveolar epithelial cells. Analysis of MAR002 using transmission electron microscopy (TEM) showed a significant presence of pili on the bacterial surface. Putative protein-coding genes involved in pili formation were identified based on the newly sequenced genome of MAR002 strain (JRHB01000001/2 or NZ_JRHB01000001/2). As assessed by qRT-PCR, the gene LH92_11085, belonging to the operon LH92_11070-11085, is overexpressed (ca. 25-fold more) in biofilm-associated cells compared to exponential planktonic cells. In the present work we investigate the role of this gene on the MAR002 biofilm phenotype. Scanning electron microscopy (SEM) and biofilm assays showed that inactivation of LH92_11085 gene significantly reduced bacterial attachment to A549 cells and biofilm formation on plastic, respectively. TEM analysis of the LH92_11085 mutant showed the absence of long pili formations normally present in the wild-type. These observations indicate the potential role this LH92_11085 gene could play in the pathobiology of A baumannii.

2-Aminoimidazoles (2AIs) have been documented to disrupt bacterial protection mechanisms, including biofilm formation and genetically encoded antibiotic resistance traits. Using Acinetobacter baumannii, we provide initial insight into the mechanism of action of a 2AI-based antibiofilm agent. Confocal microscopy confirmed that the 2AI is cell permeable, while pull-down assays identified BfmR, a response regulator that is the master controller of biofilm formation, as a target for this compound. Binding assays demonstrated specificity of the 2AI for response regulators, while computational docking provided models for 2AI-BfmR interactions. The 2AI compound studied here represents a unique small molecule scaffold that targets bacterial response regulators.

Acinetobacter baumannii is an opportunistic pathogen that causes serious infections in humans by colonizing and persisting on surfaces normally found in hospital settings. The capacity of this pathogen to persist in these settings could be due to its ability to form biofilms on inanimate surfaces. This report shows that although the ATCC 19606(T) type strain and 8 different clinical isolates form biofilms, there are significant variations in the cell density and microscopic structures of these cell aggregates, with 3 of the isolates forming pellicles floating on the surface of stagnant broth cultures. PCR indicated that, like ATCC 19606(T), all 8 clinical isolates harbor all the genetic components of the CsuA/BABCDE chaperone-usher pili assembly system, which is needed for biofilm formation on plastic. Pili detection in cells of all strains examined supports the presence and function of a pilus assembly system. However, only one of them produced the putative ATCC 19606(T) CsuA/B pilin subunit protein. Hydrophobicity tests and motility assays also showed significant variations among all tested strains and did not result in direct correlations between the biofilm phenotype and cell properties that could affect biofilm formation on abiotic surfaces. This lack of correlation among these 3 phenotypes may reflect some of the variations already reported with this pathogen, which may pose a challenge in the treatment of the infections this pathogen causes in humans using biofilm formation on abiotic surfaces as a target.

The ability of Acinetobacter baumannii to adhere to and persist on surfaces as biofilms could be central to its pathogenicity. The production of pili and a biofilm-associated protein and the expression of antibiotic resistance are needed for robust biofilm formation on abiotic and biotic surfaces. This multistep process also depends on the expression of transcriptional regulatory functions, some of which could sense nutrients available to cells. This report extends previous observations by showing that although outer membrane protein A (OmpA) of A. baumannii 19606 plays a partial role in the development of robust biofilms on plastic, it is essential for bacterial attachment to Candida albicans filaments and A549 human alveolar epithelial cells. In contrast to abiotic surfaces, the interaction with biotic surfaces is independent of the CsuA/BABCDE-mediated pili. The interaction of A. baumannii 19606 with fungal and epithelial cells also results in their apoptotic death, a response that depends on the direct contact of bacteria with these two types of eukaryotic cells. Furthermore, the bacterial adhesion phenotype correlates with the ability of bacteria to invade A549 epithelial cells. Interestingly, the killing activity of cell-free culture supernatants proved to be protease and temperature sensitive, suggesting that its cytotoxic activity is due to secreted proteins, some of which are different from OmpA.

Acinetobacter baumannii is a Gram-negative opportunistic nosocomial pathogen. This microorganism survives in hospital environments despite unfavorable conditions such as desiccation, nutrient starvation and antimicrobial treatments. It is hypothesized that its ability to persist in these environments, as well as its virulence, is a result of its capacity to form biofilms. A. baumannii forms biofilms on abiotic surfaces such as polystyrene and glass as well as biotic surfaces such as epithelial cells and fungal filaments. Pili assembly and production of the Bap surface-adhesion protein play a role in biofilm initiation and maturation after initial attachment to abiotic surfaces. Furthermore, the adhesion and biofilm phenotypes of some clinical isolates seem to be related to the presence of broad-spectrum antibiotic resistance. The regulation of the formation and development of these biofilms is as diverse as the surfaces on which this bacterium persists and as the cellular components that participate in this programmed multistep process. The regulatory processes associated with biofilm formation include sensing of bacterial cell density, the presence of different nutrients and the concentration of free cations available to bacterial cells. Some of these extracellular signals may be sensed by two-component regulatory systems such as BfmRS. This transcriptional regulatory system activates the expression of the usher-chaperone assembly system responsible for the production of pili, needed for cell attachment and biofilm formation on polystyrene surfaces. However, such a system is not required for biofilm formation on abiotic surfaces when cells are cultured in chemically defined media. Interestingly, the BfmRS system also controls cell morphology under particular culture conditions.

Acinetobacter baumannii forms biofilms on abiotic surfaces, a phenotype that may explain its ability to survive in nosocomial environments and to cause device-related infections in compromised patients. The biofilm proficiency of the 19606 type strain depends on the production of pili, cell-surface appendages assembled via the CsuAB-A-B-C-D-E chaperone-usher secretion system. The screening of a bank of isogenic insertion derivatives led to the identification of a biofilm-deficient derivative in which a transposon insertion disrupted a gene predicted to encode the response regulator of a two-component regulatory system. This gene, which was named bfmR, is required for the expression of the Csu pili chaperone-usher assembly system. This coding region is followed by an ORF encoding a putative sensor kinase that was named bfmS, which plays a less relevant role in biofilm formation when cells are cultured in rich medium. Further examination showed that the bfmR mutant was capable of attaching to abiotic surfaces, although to levels significantly lower than those of the parental strain, when it was cultured in a chemically defined minimal medium. Additionally, the morphology of planktonic cells of this mutant, when grown in minimal medium, was drastically affected, while adherent mutant cells were indistinguishable in shape and size from the parental strain. Together, these results indicate that BfmR is part of a two-component regulatory system that plays an important role in the morphology of A. baumannii 19606 cells and their ability to form biofilms on abiotic surfaces.

Biofilm formation in 92 unrelated strains of Acinetobacter baumannii isolated in a multicentre cohort study was investigated using a microtitre plate assay. Fifty-six (63%) isolates formed biofilm. These isolates were less frequently resistant to imipenem or ciprofloxacin than were non-biofilm-forming isolates (25% vs. 47%, p 0.04; and 66% vs. 94%, p 0.004, respectively). All catheter-related urinary or bloodstream infections and the sole case of shunt-related meningitis were caused by biofilm-forming strains. Multivariate analysis revealed that treatment in an intensive care unit, ciprofloxacin resistance and isolation from a respiratory sample were associated with non-biofilm-forming isolates, while previous aminoglycoside use was associated with biofilm-forming isolates.

Actinobacillus actinomycetemcomitans, a pathogen associated with oral and extra-oral infections, requires iron to grow under limiting conditions. Although incapable of producing siderophores, this pathogen could acquire iron by direct interaction with compounds such as haemin, haemoglobin, lactoferrin and transferrin. In this work the ability of different A. actinomycetemcomitans strains to bind and use different iron sources was tested. None of the strains tested used haemoglobin, lactoferrin or transferrin as sole sources of iron. However, all of them used FeCl(3) and haemin as iron sources under chelated conditions. Dot-blot binding assays showed that all strains bind lactoferrin, haemoglobin and haemin, but not transferrin. Insertion inactivation of hmsF, which encodes a predicted cell-envelope protein related to haemin-storage proteins produced by other pathogens, reduced haemin and Congo red binding drastically without affecting haemin utilization as an iron source under chelated conditions. Biofilm assays showed that all strains tested attached to and formed biofilms on plastic under iron-rich and iron-chelated conditions. However, scanning electron microscopy showed that smooth strains formed simpler biofilms than rough isolates. Furthermore, the incubation of rough cells in the presence of FeCl(3) or haemin resulted in the formation of more aggregates and microcolonies compared with the fewer cell aggregates formed when cells were grown in the presence of the synthetic iron chelator dipyridyl. These cell responses to changes in extracellular iron concentrations may reflect those that this pathogen expresses under the conditions it encounters in the human oral cavity.

Acinetobacter baumannii causes severe infections in compromised patients, survives on abiotic surfaces in hospital environments and colonizes different medical devices. In this study the analysis of the processes involved in surface attachment and biofilm formation by the prototype strain 19606 was initiated. This strain attaches to and forms biofilm structures on plastic and glass surfaces, particularly at the liquid-air interface of cultures incubated stagnantly. The cell aggregates, which contain cell stacks separated by water channels, formed under different culture conditions and were significantly enhanced under iron limitation. Electron and fluorescence microscopy showed that pili and exopolysaccharides are part of the cell aggregates formed by this strain. Electron microscopy of two insertion derivatives deficient in attachment and biofilm formation revealed the disappearance of pili-like structures and DNA sequencing analysis showed that the transposon insertions interrupted genes with the highest similarity to hypothetical genes found in Pseudomonas aeruginosa, Pseudomonas putida and Vibrio parahaemolyticus. Although the products of these genes, which have been named csuC and csuE, have no known functions, they are located within a polycistronic operon that includes four other genes, two of which encode proteins related to chaperones and ushers involved in pili assembly in other bacteria. Introduction of a copy of the csuE parental gene restored the adherence phenotype and the presence of pili on the cell surface of the csuE mutant, but not that of the csuC derivative. These results demonstrate that the expression of a chaperone-usher secretion system, some of whose components appear to be acquired from unrelated sources, is required for pili formation and the concomitant attachment to plastic surfaces and the ensuing formation of biofilms by A. baumannii cells.