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Background Acinetobacter baumannii causes a wide range of dangerous infections due to the emergence of pandrug-resistant strains. Therefore, there is a need for alternative therapeutics to treat these infections, including those targeting the host immune responses. However, immune responses, especially the humoral response against this pathogen, are poorly understood. Methods This study investigated the lymphocyte-mediated innate immune resistance to A. baumannii AB5075 pulmonary infection using B- and T-cell–deficient (Rag2−/−) mice, the protective effect of natural antibodies (NAbs), and the expression of complement-mediated responses using a mouse pneumonia model. Results Our results showed that intranasally infected Rag2−/− mice are impaired in clearing bacteria from lung, liver, and spleen at 24 hours postinfection compared to wildtype mice. Animal pretreatment with normal mouse serum or purified antibodies from naive mice rescued Rag2−/− mice from infection. Analysis of C3 complement protein binding demonstrated that NAbs increased C3 protein deposition on A. baumannii cells, indicating the activation of the classical complement pathway by NAbs. Conclusions Overall, our study shows that NAbs mediate innate immune resistance against A. baumannii, a finding that may lead to the development of effective therapies against human infections caused by this antibiotic-resistant A. baumannii.

Acinetobacter baumannii is a multidrug-resistant pathogen that causes numerous infections associated with high mortality rates. Exposure to human body fluids, such as human pleural fluid (HPF) and human serum, modulates gene expression in A. baumannii, leading to changes in its pathogenic behavior. Diverse degrees of effects at the transcriptional level were observed in susceptible and carbapenem-resistant strains. The transcriptional analysis of AB5075, a hyper-virulent and extensively drug-resistant strain showed changes in genes associated with quorum sensing, quorum quenching, fatty acids metabolism, and high-efficient iron uptake systems. In addition, the distinctive role of human serum albumin (HSA) as a critical component of HPF was evidenced. In the present work, we used model strain to analyze more deeply into the contribution of HSA in triggering A. baumannii's response. By qRT-PCR analysis, changes in the expression level of genes associated with quorum sensing, biofilm formation, and phenylacetic acid pathway were observed. Phenotypic approaches confirmed the transcriptional response. HSA, a predominant component of HPF, can modulate the expression and behavior of genes not only in a hyper-virulent and extensively drug-resistant A. baumannii model, but also in other strains with a different degree of susceptibility and pathogenicity.

The opportunistic pathogen A. baumannii has a remarkable capacity to persist in the hospital environment and cause devastating human infections. This capacity can be attributed partly to the sensing and regulatory systems that enable this pathogen to modify its physiology based on environmental cues. One of the signals that A. baumannii senses and responds to is light through the sensing and regulatory roles of the BlsA photoreceptor protein in cells cultured at temperatures below 30°C. This report presents evidence that a light stimulon is operational at 37°C, a condition at which the BlsA production and activity are drastically impaired. Global transcriptional analysis showed that the 37°C light stimulon includes the differential expression of chromosomal genes encoding a wide range of functions that are known to be involved in the adaptation to different metabolic conditions, as well as virulence and persistence in the host and the medical environment. Unexpectedly, the 37°C light stimulon also includes the differential expression of conjugation functions encoded by pAB3 plasmid genes. Our work further demonstrates that the TetR1 and H-NS regulators encoded by this conjugative plasmid control the expression of H(2)O(2) resistance and surface motility, respectively. Furthermore, our data showed that pAB3 has an overall negative effect on the expression of these phenotypes and plays no significant virulence role. Although the nature of the bacterial factors and the mechanisms by which the regulation is attained at 37°C remain unknown, taken together, our work expands the current knowledge about light sensing and gene regulation in A. baumannii. IMPORTANCE As a facultative pathogen, Acinetobacter baumannii persists in various environments by sensing different environmental cues, including light. This report provides evidence of light-dependent regulation at 37°C of the expression of genes coding for a wide range of functions, including those involved in the conjugation of the pAB3 plasmid. Although this plasmid affects the expression of virulence traits when tested under laboratory conditions, it does not have a significant impact when tested using ex vivo and in vivo experimental models. These findings provide a better understanding of the interplay between light regulation and plasmid persistence in the pathobiology of A. baumannii.

Cefiderocol (CFDC) is a novel chlorocatechol-substituted siderophore antibiotic approved to treat complicated urinary tract infections (cUTI) and hospital-acquired and ventilator-acquired pneumonia (HAP/VAP). Previous work determined that albumin-rich human fluids increase the minimum inhibitory concentration (MICs) of Acinetobacter baumannii against CFDC and reduce the expression of genes related to iron uptake systems. This latter effect may contribute to the need for higher concentrations of CFDC to inhibit growth. The presence of human urine (HU), which contains low albumin concentrations, did not modify MIC values of two carbapenem-resistant A. baumannii. Levels of resistance to CFDC were not modified by HU in strain AMA40 but were reduced in strain AB5075. Expanding the studies to other carbapenem-resistant A. baumannii isolates showed that the presence of HU resulted in unmodified or reduced MIC of CDFC values. The expression of piuA, pirA, bauA, and bfnH determined by qRT-PCR was enhanced in A. baumannii AMA40 and AB5075 by the presence of HU in the culture medium. All four tested genes code for functions related to recognition and transport of ferric-siderophore complexes. The effect of HU on expression of pbp1, pbp3, blaOXA-51-like, blaADC, and blaNDM-1, genes associated with resistance to β-lactams, as well as genes coding for efflux pumps and porins was variable, showing dependence with the strain analyzed. We conclude that the lack of significant concentrations of albumin and free iron in HU makes this fluid behave differently from others we tested. Unlike other albumin rich fluids, the presence of HU does not impact the antibacterial activity of CFDC when tested against A. baumannii.

Acinetobacter baumannii is a catalase-positive Gram-negative bacterial pathogen that causes severe infections among compromised patients. Among its noteworthy regulatory mechanisms, this microorganism regulates its lifestyle through the blue light using flavin (BLUF) protein BlsA. This protein regulates a diverse set of cellular processes that include, but are not limited to, motility, biofilm formation, phenylacetic acid metabolism, iron uptake, and catalase activity. We set out to determine how A. baumannii regulates catalase activity and other related oxidative stress phenotypes in response to light. Notably, because A. baumannii ATCC 17978 encodes four catalase homologs – which we refer to as KatA, KatE, KatE2, and KatG – we also aimed to show which of these enzymes exhibit light- and BlsA-dependent activity. Our work not only provides insight into the general function of all four catalase homologs and the impact of light on these functions, but also directly identifies KatE as a BlsA-regulated enzyme. We further demonstrate that the regulation of KatE by BlsA is dependent on a lysine residue that we previously demonstrated to be necessary for the regulation of surface motility. Furthermore, we show that BlsA’s five most-C-terminal residues – previously considered dispensable for BlsA’s overall function – are necessary for the light-independent and light-dependent regulation of catalase and superoxide dismutase activities, respectively. We hypothesize that these identified critical residues are necessary for BlsA’s interaction with protein partners including the transcriptional regulators Fur and BfmR. Together these data expand the understanding regarding how A. baumannii uses light as a signal to control oxidative stress resistance mechanisms that are critical for its pathophysiology.

Cefiderocol (CFDC) is a novel chlorocatechol-substituted siderophore cephalosporin, approved by the Food and Drug Administration (FDA) for treating complicated urinary tract infections and for hospital-acquired and ventilator-acquired pneumonia. In previous work, human serum, human serum albumin,HSA, and human pleural fluid, HPF, were shown to increase the minimum inhibitory concentration (MICs) of Acinetobacter baumannii against CFDC. These fluids are also associated with a reduction in the expression of genes related to iron uptake systems, which could explain the need for higher concentrations of CFDC to exert inhibitory action. Herein, we analyzed the impact of human urine (HU), which contains low albumin concentrations, on the expression of iron-uptake related genes and MIC values of two carbapenem-resistant A. baumannii. Levels of resistance to CFDC were not modified by HU in strain AMA40 but were reduced in the case of strain AB5075. Testing other carbapenem-resistant A. baumannii isolates showed that the CFDC MICs were unmodified or reduced in the presence of HU. The expression of piuA, pirA, bauA, and bfnH determined by qRT-PCR was enhanced in both strains when HU was present in the culture medium. All four tested genes are involved in recognizing ferric siderophore complexes or internalization into the cell’s cytosol. In contrast, the effect of HU on genes associated with resistance to β-lactams, antibiotics commonly used to treat urinary tract infections caused by A. baumannii, was variable; the transcriptional analysis of pbp1, pbp3, blaOXA−51−like, blaADC, and blaNDM−1 showed significant variation. In summary, HU, probably due to the albumin and free iron content, does not adversely impact or slightly improves the activity of CFDC when tested against A. baumannii in urine in contrast to other human bodily fluids.

Acinetobacter baumannii is a common nosocomial pathogen that utilizes numerous mechanisms to aid its survival in both the environment and the host. Coordination of such mechanisms requires an intricate regulatory network. We report here that A. baumannii can directly regulate several stress-related pathways via the two-component regulatory system BfmRS. Similar to previous studies, results from transcriptomic analysis showed that mutation of the BfmR response regulator causes dysregulation of genes required for the oxidative stress response, the osmotic stress response, the misfolded protein/heat shock response, Csu pilus/fimbria production, and capsular polysaccharide biosynthesis. We also found that the BfmRS system is involved in controlling siderophore biosynthesis and transport, and type IV pili production. We provide evidence that BfmR binds to various stress-related promoter regions and show that BfmR alone can directly activate transcription of some stress-related genes. Additionally, we show that the BfmS sensor kinase acts as a BfmR phosphatase to negatively regulate BfmR activity. This work highlights the importance of the BfmRS system in promoting survival of A. baumannii. IMPORTANCE Acinetobacter baumannii is a nosocomial pathogen that has extremely high rates of multidrug resistance. This organism’s ability to endure stressful conditions is a key part of its ability to spread in the hospital environment and cause infections. Unlike other members of the gammaproteobacteria, A. baumannii does not encode a homolog of the RpoS sigma factor to coordinate its stress response. Here, we demonstrate that the BfmRS two-component system directly controls the expression of multiple stress resistance genes. Our findings suggest that BfmRS is central to a unique scheme of general stress response regulation by A. baumannii.

Cefiderocol is a siderophore antibiotic that co-opts iron transporters to facilitate cell entry. We show that genes related to iron uptake systems and resistance to β-lactams in Acinetobacter baumannii have altered expression levels in the presence of human serum, human serum albumin, or human pleural fluid. Cefiderocol MICs are also raised in the presence of the mentioned fluids. Clinical response in A. baumannii infections may be related to the interplay of these human factors.

BackgroundNew strategies are needed to slow the emergence of antibiotic resistance among bacterial pathogens. In particular, society is experiencing a crisis of antibiotic-resistant infections caused by Gram-negative bacterial pathogens and novel therapeutics are desperately needed to combat such diseases. Acquisition of iron from the host is a nearly universal requirement for microbial pathogens—including Gram-negative bacteria—to cause infection. We have previously reported that apo-transferrin (lacking iron) can inhibit the growth of Staphylococcus aureus in culture and diminish emergence of resistance to rifampicin.ObjectivesTo define the potential of apo-transferrin to inhibit in vitro growth of Klebsiella pneumoniae and Acinetobacter baumannii, key Gram-negative pathogens, and to reduce emergence of resistance to antibiotics.MethodsThe efficacy of apo-transferrin alone or in combination with meropenem or ciprofloxacin against K. pneumoniae and A. baumannii clinical isolates was tested by MIC assay, time–kill assay and assays for the selection of resistant mutants.ResultsWe confirmed that apo-transferrin had detectable MICs for all strains tested of both pathogens. Apo-transferrin mediated an additive antimicrobial effect for both antibiotics against multiple strains in time–kill assays. Finally, adding apo-transferrin to ciprofloxacin or meropenem reduced the emergence of resistant mutants during 20 day serial passaging of both species.ConclusionsThese results suggest that apo-transferrin may have promise to suppress the emergence of antibiotic-resistant mutants when treating infections caused by Gram-negative bacteria.

Acinetobacter baumannii has been recognized as a critical pathogen that causes severe infections worldwide not only because of the emergence of extensively drug-resistant (XDR) derivatives, but also because of its ability to persist in medical environments and colonize compromised patients. While there are numerous reports describing the mechanisms by which this pathogen acquires resistance genes, little is known regarding A. baumannii’s virulence functions associated with rare manifestations of infection such as necrotizing fasciitis, making the determination and implementation of alternative therapeutic targets problematic. To address this knowledge gap, this report describes the analysis of the NFAb-1 and NFAb-2 XDR isolates, which were obtained at two time points during a fatal case of necrotizing fasciitis, at the genomic and functional levels. The comparative genomic analysis of these isolates with the ATCC 19606T and ATCC 17978 strains showed that the NFAb-1 and NFAb-2 isolates are genetically different from each other as well as different from the ATCC 19606T and ATCC 17978 clinical isolates. These genomic differences could be reflected in phenotypic differences observed in these NFAb isolates. Biofilm, cell viability and flow cytometry assays indicate that all tested strains caused significant decreases in A549 human alveolar epithelial cell viability with ATCC 17978, NFAb-1 and NFAb-2 producing significantly less biofilm and significantly more hemolysis and capacity for intracellular invasion than ATCC 19606T. NFAb-1 and NFAb-2 also demonstrated negligible surface motility but significant twitching motility compared to ATCC 19606T and ATCC 17978, likely due to the presence of pili exceeding 2 µm in length, which are significantly longer and different from those previously described in the ATCC 19606T and ATCC 17978 strains. Interestingly, infection with cells of the NFAb-1 isolate, which were obtained from a premortem blood sample, lead to significantly higher mortality rates than NFAb-2 bacteria, which were obtained from postmortem tissue samples, when tested using the Galleria mellonella in vivo infection model. These observations suggest potential changes in the virulence phenotype of the A. baumannii necrotizing fasciitis isolates over the course of infection by mechanisms and cell processes that remain to be identified.

Acinetobacter baumannii is a nosocomial pathogen capable of causing serious infections associated with high rates of morbidity and mortality. Due to its antimicrobial drug resistance profile, A. baumannii is categorized as an urgent priority pathogen by the Centers for Disease Control and Prevention in the United States and a priority group 1 critical microorganism by the World Health Organization. Understanding how A. baumannii adapts to different host environments may provide critical insights into strategically targeting this pathogen with novel antimicrobial and biological therapeutics. Exposure to human fluids was previously shown to alter the gene expression profile of a highly drug-susceptible A. baumannii strain A118 leading to persistence and survival of this pathogen. Herein, we explore the impact of human pleural fluid (HPF) and human serum albumin (HSA) on the gene expression profile of a highly multi-drug-resistant strain of A. baumannii AB5075. Differential expression was observed for ~30 genes, whose products are involved in quorum sensing, quorum quenching, iron acquisition, fatty acid metabolism, biofilm formation, secretion systems, and type IV pilus formation. Phenotypic and further transcriptomic analysis using quantitative RT-PCR confirmed RNA-seq data and demonstrated a distinctive role of HSA as the molecule involved in A. baumannii’s response.

Acinetobacter baumannii is a nosocomial pathogen capable of causing serious infections associated with high rates of morbidity and mortality. Due to its antimicrobial drug resistance profile, A. baumannii is categorized as an urgent priority pathogen by the Centers for Disease Control and Prevention in the United States and priority group 1 critical microorganism by the World Health Organization. Understanding how A. baumannii adapts to different host environments may provide critical insights into strategically targeting this pathogen with novel antimicrobial and biological therapeutics. Exposure to human fluids was previously shown to alter the gene expression profile of a highly drug susceptible A. baumannii strain A118 leading to persistence and survival of this pathogen. Herein, we explore the impact of human pleural fluid (HPF) and human serum albumin (HSA) on the gene expression profile of a highly multi-drug resistant strain of A. baumannii AB5075. Differential expression was observed for ~30 genes, whose products are involved in quorum sensing, quorum quenching, iron acquisition, fatty acid metabolism, biofilm formation, secretion systems and type IV pilus formation. Phenotypic and further transcriptomic analysis using quantitative RT-PCR confirmed RNA-seq data and pointed out a distinctive role of HSA as the molecule involved in A. baumannii response.

Multi-drug resistant (MDR) Acinetobacter baumannii (Ab) and Acinetobacter spp. present monumental global health challenges. These organisms represent model Gram-negative pathogens with known antibiotic resistance and biofilm-forming properties. Herein, a novel, nontoxic biocide, AB569, consisting of acidified nitrite (A-NO2-) and ethylenediaminetetraacetic acid (EDTA), demonstrated bactericidal activity against all Ab and Acinetobacter spp. strains, respectively. Average fractional inhibitory concentrations (FICs) of 0.25 mM EDTA plus 4 mM A-NO2- were observed across several clinical reference and multiple combat wound isolates from the Iraq/Afghanistan wars. Importantly, toxicity testing on human dermal fibroblasts (HDFa) revealed an upper toxicity limit of 3 mM EDTA plus 64 mM A-NO2-, and thus are in the therapeutic range for effective Ab and Acinetobacter spp. treatment. Following treatment of Ab strain ATCC 19606 with AB569, quantitative PCR analysis of selected genes products to be responsive to AB569 revealed up-regulation of iron regulated genes involved in siderophore production, siderophore biosynthesis non-ribosomal peptide synthetase module (SBNRPSM), and siderophore biosynthesis protein monooxygenase (SBPM) when compared to untreated organisms. Taken together, treating Ab infections with AB569 at inhibitory concentrations reveals the potential clinical application of preventing Ab from gaining an early growth advantage during infection followed by extensive bactericidal activity upon subsequent exposures.

Acinetobacter baumannii has been recognized as a critical pathogen that causes severe infections worldwide not only because of the emergence of extensively drug-resistant (XDR) derivatives, but also because of its ability to persist in medical environments and colonize compromised patients. While there are numerous reports describing the mechanisms by which this pathogen acquires resistance genes, little is known regarding A. baumannii’s virulence functions associated with rare manifestations of infection such as necrotizing fasciitis, making the determination and implementation of alternative therapeutic targets problematic. To address this knowledge gap, this report describes the analysis of the NFAb-1 and NFAb-2 XDR isolates, which were obtained at two time points during a fatal case of necrotizing fasciitis, at the genomic and functional levels. The comparative genomic analysis of these isolates with the ATCC 19606T and ATCC 17978 strains showed that the NFAb-1 and NFAb-2 isolates are genetically different from each other as well as different from the ATCC 19606T and ATCC 17978 clinical isolates. These genomic differences could be reflected in phenotypic differences observed in these NFAb isolates. Biofilm, cell viability and flow cytometry assays indicate that all tested strains caused significant decreases in A549 human alveolar epithelial cell viability with ATCC 17978, NFAb-1 and NFAb-2 producing significantly less biofilm and significantly more hemolysis and capacity for intracellular invasion than ATCC 19606T. NFAb-1 and NFAb-2 also demonstrated negligible surface motility but significant twitching motility compared to ATCC 19606T and ATCC 17978, likely due to the presence of pili exceeding 2 µm in length, which are significantly longer and different from those previously described in the ATCC 19606T and ATCC 17978 strains. Interestingly, infection with cells of the NFAb-1 isolate, which were obtained from a premortem blood sample, lead to significantly higher mortality rates than NFAb-2 bacteria, which were obtained from postmortem tissue samples, when tested using the Galleria mellonella in vivo infection model. These observations suggest potential changes in the virulence phenotype of the A. baumannii necrotizing fasciitis isolates over the course of infection by mechanisms and cell processes that remain to be identified.

Antibiotic-resistant bacteria are an increasing and serious health concern worldwide, and multidrug-resistant pathogens are increasingly emerging among patients across the United States. Researchers are exploring sources of traditional medicines, including mushrooms, to find new antibiotic compounds. In this study, the antibiotic activities of 75 mushrooms collected in the area surrounding Oxford, Ohio (USA), were assayed for antibiotic activity against 6 bacterial strains (Pseudomonas aeruginosa reference strains PAO1 and PA14, P. fluorescens, Bacillus subtilis, Staphylococcus epidermidis, and Micrococcus luteus). Mushroom samples were identified by using DNA ribotyping. We used methanol and water extracts of mushrooms in agar diffusion assays to screen for antibiotic activity toward each bacterial strain. A total of 25 mushroom species had antibacterial activity against at least 1 bacterium. Water extracts of Polyporus squamosus, Ganoderma applanatum, Lentinellus subaustralis, Laetiporus sulphureus, G. lucidum, and Trametes versicolor exhibited strong antibiotic activity against all bacterial strains tested. Water and methanol extracts from 25 mushroom species had significant activity against most of the bacteria tested. A minimum inhibitory concentration (MIC) against S. epidermidis was determined for all samples that exhibited antibiotic activity in the disk assay. The G. lucidum and L. sulphureus extracts displayed the strongest inhibition, with an MIC of 0.1 mg/mL.

Carbapenem-resistant Acinetobacter baumannii is a bacterial pathogen with serious implications for human health and is recognized as an urgent threat by the Centers for Disease Control and Prevention (CDC). Total DNA from two A. baumannii clinical isolates collected over 3 days from a fatal case of necrotizing fasciitis has been sequenced to >30× coverage.

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.

BACKGROUND Acinetobacter baumannii has emerged as an increasingly important and successful opportunistic human pathogen due to its ability to withstand harsh environmental conditions, its characteristic virulence factors, and quick adaptability to stress. METHODS We developed a clinically relevant murine model of A. baumannii traumatic wound infection to determine the effect of local wound environment on A. baumannii virulence. Mice underwent rectus muscle crush injury combined with ischemia created by epigastric vessel ligation, followed by A. baumannii inoculation. Reiterative experiments were performed using (1) a mutant deficient in the production of the siderophore acinetobactin, or (2) iron supplementation of the wound milieu. Mice were euthanized 7 days later, and rectus muscle analyzed for signs of clinical infection, HIF1α accumulation, bacterial abundance, and colony morphotype. To determine the effect of wound milieu on bacterial virulence, Galleria mellonella infection model was used. RESULTS The combination of rectus muscle injury with ischemia and A. baumannii inoculation resulted in 100% incidence of clinical wound infection that was significantly higher compared with other groups (n = 15/group, p < 0.0001). The highest level of wound infection was accompanied by the highest level of A. baumannii colonization (p < 0.0001) and the highest degree of HIF1α accumulation (p < 0.05). A. baumannii strains isolated from injured/ischemic muscle with clinical infection displayed a rough morphotype and a higher degree of virulence as judged by G. mellonella killing assay as compared with smooth morphotype colonies isolated from injured muscle without clinical infection (100% vs. 60%, n = 30 Log-Rank test, p = 0.0422). Iron supplementation prevented wound infection (n = 30, p < 0.0001) and decreased HIF1α (p = 0.039643). Similar results of decrease in wound infection and HIF1α were obtained when A. baumannii wild type was replaced with its derivative mutant [INCREMENT]BasD deficient in acinetobactin production. CONCLUSION The ability of A. baumannii to cause infections in traumatized wound relies on its ability to scavenge iron and can be prevented by iron supplementation to the wound milieu.

Acinetobacter baumannii A118, a strain isolated from the blood of an infected patient, is naturally competent and unlike most clinical strains, is susceptible to a variety of different antibiotics including those usually used for selection in genetic manipulations. These characteristics make strain A118 a convenient model for genetic studies of A. baumannii. To identify potential virulence factors, its complete genome was analyzed and compared to other A. baumannii genomes. A. baumannii A118 includes gene clusters coding for the acinetobactin and baumannoferrin iron acquisition systems. Iron-regulated expression of the BauA outer membrane receptor for ferric-acinetobactin complexes was confirmed as well as the utilization of acinetobactin. A. baumannii A118 also possesses the feoABC genes, which code for the main bacterial ferrous uptake system. The functionality of baumannoferrin was suggested by the ability of A. baumannii A118 culture supernatants to cross feed an indicator BauA-deficient strain plated on iron-limiting media. A. baumannii A118 behaved as non-motile but included the csuA/BABCDE chaperone-usher pilus assembly operon and produced biofilms on polystyrene and glass surfaces. While a known capsular polysaccharide (K) locus was identified, the outer core polysaccharide (OC) locus, which belongs to group B, showed differences with available sequences. Our results show that despite being susceptible to most antibiotics, strain A118 conserves known virulence-related traits enhancing its value as model to study A. baumannii pathogenicity.

There has been renewed interest in combining traditional small-molecule antimicrobial agents with nontraditional therapies to potentiate antimicrobial effects. Apotransferrin, which decreases iron availability to microbes, is one such approach. We conducted a 48-h one-compartment in vitro infection model to explore the impact of apotransferrin on the bactericidal activity of ciprofloxacin. The challenge panel included four Klebsiella pneumoniae isolates with ciprofloxacin MIC values ranging from 0.08 to 32 mg/liter. Each challenge isolate was subjected to an ineffective ciprofloxacin monotherapy exposure (free-drug area under the concentration-time curve over 24 h divided by the MIC [AUC/MIC ratio] ranging from 0.19 to 96.6) with and without apotransferrin. As expected, the no-treatment and apotransferrin control arms showed unaltered prototypical logarithmic bacterial growth. We identified relationships between exposure and change in bacterial density for ciprofloxacin alone (R(2) = 0.64) and ciprofloxacin in combination with apotransferrin (R(2) = 0.84). Addition of apotransferrin to ciprofloxacin enabled a remarkable reduction in bacterial density across a wide range of ciprofloxacin exposures. For instance, at a ciprofloxacin AUC/MIC ratio of 20, ciprofloxacin monotherapy resulted in nearly 2 log(10) CFU increase in bacterial density, while the combination of apotransferrin and ciprofloxacin resulted in 2 log(10) CFU reduction in bacterial density. Furthermore, addition of apotransferrin significantly reduced the emergence of ciprofloxacin-resistant subpopulations compared to monotherapy. These data demonstrate that decreasing the rate of bacterial replication with apotransferrin in combination with antimicrobial therapy represents an opportunity to increase the magnitude of the bactericidal effect and to suppress the growth rate of drug-resistant subpopulations.

Acinetobacter baumannii A118, a strain isolated from the blood of an infected patient, is naturally competent and unlike most clinical strains, is susceptible to a variety of different antibiotics including those usually used for selection in genetic manipulations. These characteristics make strain A118 a convenient model for genetic studies of A. baumannii. To identify potential virulence factors, its complete genome was analyzed and compared to other A. baumannii genomes. A. baumannii A118 includes gene clusters coding for the acinetobactin and baumannoferrin iron acquisition systems. Iron-regulated expression of the BauA outer membrane receptor for ferric-acinetobactin complexes was confirmed as well as the utilization of acinetobactin. A. baumannii A118 also possesses the feoABC genes, which code for the main bacterial ferrous uptake system. The functionality of baumannoferrin was suggested by the ability of A. baumannii A118 culture supernatants to cross feed an indicator BauA-deficient strain plated on iron-limiting media. A. baumannii A118 behaved as non-motile but included the csuA/BABCDE chaperone-usher pilus assembly operon and produced biofilms on polystyrene and glass surfaces. While a known capsular polysaccharide (K) locus was identified, the outer core polysaccharide (OC) locus, which belongs to group B, showed differences with available sequences. Our results show that despite being susceptible to most antibiotics, strain A118 conserves known virulence-related traits enhancing its value as model to study A. baumannii pathogenicity.

Siderophores are high-affinity iron chelators produced and used by bacteria to prosper under iron-limiting conditions they normally encounter in the environment and hosts. In this chapter, we describe the isolation and purification of the siderophores acinetobactin and baumannoferrin produced by the bacterial pathogen Acinetobacter baumannii using XAD-7 batch adsorption and high-pressure liquid chromatography (HPLC). We also describe chemical tests and biological assays used to detect the presence of catechol and hydroxamate siderophores in culture supernatants, XAD-7 extracts, and HPLC fractions.

16 pages, 1 table, 8 figures.-- A. Pérez ... et al., Acinetobacter baumannii is a hospital-acquired pathogen that shows an extraordinary capacity to stay in the hospital environment. Adherence of the bacteria to eukaryotic cells or to abiotic surfaces is the first step for establishing an infection. The A. baumannii strain AbH12O-A2 showed an exceptional ability to adhere to A549 epithelial cells. The AbFhaB/FhaC 2-partner secretion (TPS) system involved in adhesion was discovered after the screening of the recently determined A. baumannii AbH12O-A2 strain genome (CP009534.1). The AbFhaB is a large exoprotein which transport to the bacterial surface is mediated by the AbFhaC protein. In the present study, the role of this TPS system in the AbH12O-A2 adherence phenotype was investigated. The functional inactivation of this 2-partner secretion system was addressed by analyzing the outer membrane vesicles (OMV) proteomic profile from the wild-type strain and its derivative mutant AbH12O-A2ΔfhaC demonstrating that AbFhaB is no longer detected in the absence of AbFhaC. Scanning electron microscopy (SEM) and adhesion experiments demonstrated that inactivation of the AbFhaB/FhaC system significantly decreases bacterial attachment to A549 alveolar epithelial cells. Moreover, it has been demonstrated that this 2-partner secretion system is involved in fibronectin-mediated adherence of the A. baumannii AbH12O-A2 isolate. Finally, we report that the AbFhaB/FhaC system is involved in virulence when tested using invertebrate and vertebrate hosts. These data suggest the potential role that this AbFhaB/FhaC secretion system could play in the pathobiology of A. baumannii, This work has been funded by Projects PI12/00552 and PI15/00860 to GB, PI11/01034 to MP, CP13/00226 to AB, and P14/000059 to MP and AB, all integrated in the National Plan for Scientific Research, Development and Technological Innovation 2013–2016 and funded by the ISCIII - General Subdirection of Assessment and Promotion of the Research – European Regional Development Fund (FEDER) “A way of making Europe.” Miami University Research Funds from the department of Microbiology supported this work. We also want to thank the Spanish Network for Research in Infectious Diseases (REIPI RD12/0015/0014 to GB), co-financed by the European Development Regional Fund (EDRF) “A Way to Achieve Europe, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad as well as Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC). A.P. was financially supported by the Galician Plan for Research, Innovation and Growth (I2C Plan 2012–2016). J. A. Vallejo was financially supported by the Sara Borrell Program (ISCIII, Spain CD13/00373). S. Rumbo-Feal was financially supported by the Agustí Pumarola Grant (Societat Catalana de Malalties Infeccioses i Microbiologia Clínica, SCMIMC) and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC). Finally, we thank Chua Kim Lee (University of Singapore) for providing pMo130-TelR.

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., The results of this work have been funded by the Projects PI11/01034 to MP and PI14/00059 to MP and AB and PI12/00552 and PI15/00860 to GB, integrated in the National Plan for Scientific Research, Development and Technological Innovation 2013-2016 and funded by the ISCIII- General Subdirection of Assessment and Promotion of the Research – European Regional Development Fund (FEDER) “A way of making Europe” and Miami University (Ohio, USA) Research Funds. We also thank Spanish Network for Research in Infectious Diseases (REIPI RD12/0015/0014 to GB), cofinanced by the European Development Regional Fund (EDRF) “A Way to Achieve Europe, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad. A.P. was financially supported by the Galician Plan for Research, Innovation and Growth (I2C Plan 2012-2016). J.A. Vallejo was financially supported by the Sara Borrell Program (ISCIII, Spain CD13/00373). We wish to thank grant GEIH/REIPI-Ab2010 from Instituto de Salud Carlos III - Ministerio Economía y Competitividad for providing MAR002 strain and M.I. Voskuil for pMo130.

Stagnation in antimicrobial development has led to a serious threat to public health because some Acinetobacter baumannii infections have become untreatable. New therapeutics with alternative mechanisms of action to combat A. baumannii are therefore necessary to treat these infections. To this end, the virulence of A. baumannii isolates with various antimicrobial susceptibilities was assessed when the isolates were treated with miltefosine, a phospholipase C inhibitor. Phospholipase C activity is a contributor to A. baumannii virulence associated with hemolysis, cytolysis of A549 human alveolar epithelial cells, and increased mortality in the Galleria mellonella experimental infection model. While the effects on bacterial growth were variable among strains, miltefosine treatment significantly reduced both the hemolytic and cytolytic activity of all treated A. baumannii strains. Additionally, scanning electron microscopy of polarized A549 cells infected with bacteria of the A. baumannii ATCC 19606T strain or the AB5075 multidrug-resistant isolate showed a decrease in A549 cell damage with a concomitant increase in the presence of A549 surfactant upon administration of miltefosine. The therapeutic ability of miltefosine was further supported by the results of G. mellonella infections, wherein miltefosine treatment of animals infected with ATCC 19606T significantly decreased mortality. These data demonstrate that inhibition of phospholipase C activity results in the overall reduction of A. baumannii virulence in both in vitro and in vivo models, making miltefosine a viable option for the treatment of A. baumannii infections, particularly those caused by multidrug-resistant isolates.

Siderophores are high-affinity iron chelators produced and used by bacteria to prosper under iron-limiting conditions they normally encounter in the environment and hosts. In this chapter, we describe the isolation and purification of the siderophores acinetobactin and baumannoferrin produced by the bacterial pathogen Acinetobacter baumannii using XAD-7 batch adsorption and high-pressure liquid chromatography (HPLC). We also describe chemical tests and biological assays used to detect the presence of catechol and hydroxamate siderophores in culture supernatants, XAD-7 extracts, and HPLC fractions.

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 photoregulated pilus 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.

Stagnation in antimicrobial development has led to a serious threat to public health because some Acinetobacter baumannii infections have become untreatable. New therapeutics with alternative mechanisms of action to combat A. baumannii are therefore necessary to treat these infections. To this end, the virulence of A. baumannii isolates with various antimicrobial susceptibilities was assessed when the isolates were treated with miltefosine, a phospholipase C inhibitor. Phospholipase C activity is a contributor to A. baumannii virulence associated with hemolysis, cytolysis of A549 human alveolar epithelial cells, and increased mortality in the Galleria mellonella experimental infection model. While the effects on bacterial growth were variable among strains, miltefosine treatment significantly reduced both the hemolytic and cytolytic activity of all treated A. baumannii strains. Additionally, scanning electron microscopy of polarized A549 cells infected with bacteria of the A. baumannii ATCC 19606(T) strain or the AB5075 multidrug-resistant isolate showed a decrease in A549 cell damage with a concomitant increase in the presence of A549 surfactant upon administration of miltefosine. The therapeutic ability of miltefosine was further supported by the results of G. mellonella infections, wherein miltefosine treatment of animals infected with ATCC 19606(T) significantly decreased mortality. These data demonstrate that inhibition of phospholipase C activity results in the overall reduction of A. baumannii virulence in both in vitro and in vivo models, making miltefosine a viable option for the treatment of A. baumannii infections, particularly those caused by multidrug-resistant isolates.

Acinetobacter baumannii adapts to different environmental conditions by expressing complex regulatory circuitry. Recent studies revealed that this circuitry includes regulatory factors that control the emergence of distinct bacterial subpopulations, which are critical for the capacity of this pathogen to persist in medical settings and cause infections in compromised hosts.

Multiresistant Acinetobacter baumannii , a common etiologic agent of severe nosocomial infections in compromised hosts, usually harbors aac(6 ′ )-Ib. This gene specifies resistance to amikacin and other aminoglycosides, seriously limiting the effectiveness of these antibiotics. An antisense oligodeoxynucleotide (ODN4) that binds to a duplicated sequence on the aac(6 ′ )-Ib mRNA, one of the copies overlapping the initiation codon, efficiently inhibited translation in vitro . An isosequential nuclease-resistant hybrid oligomer composed of 2′,4′-bridged nucleic acid-NC (BNA NC ) residues and deoxynucleotides (BNA NC -DNA) conjugated to the permeabilizing peptide (RXR) 4 XB (“X” and “B” stand for 6-aminohexanoic acid and β-alanine, respectively) (CPPBD4) inhibited translation in vitro at the same levels observed in testing ODN4. Furthermore, CPPBD4 in combination with amikacin inhibited growth of a clinical A. baumannii strain harboring aac(6 ′ )-Ib in liquid cultures, and when both compounds were used as combination therapy to treat infected Galleria mellonella organisms, survival was comparable to that seen with uninfected controls.

Acinetobacter baumannii AYE does not produce acinetobactin but grows under iron limitation. Accordingly, analyses of AYE iron-restricted culture supernatants resulted in the isolation of two fractions, which contained only hydroxamates and showed siderophore activity. Structural analyses identified baumannoferrin A and baumannoferrin B, which differ only by a double bond. These siderophores are composed of citrate, 1,3-diaminopropane, 2,4-diaminobutyrate, decenoic acid, and α-ketoglutarate. Analysis of the AYE genome showed the presence of a 12-gene cluster coding for proteins similar to those involved in the production and utilization of the hydroxamate siderophores acinetoferrin and achromobactin. As A. baumannii AYE does not produce acinetobactin and harbors only one gene cluster encoding the production and utilization of a siderophore, this strain's growth under iron limitation depends on baumannoferrin, a novel hydroxamate that could play a role in its virulence.

The capacity of Acinetobacter baumannii to persist and cause infections depends on its interaction with abiotic and biotic surfaces, including those found on medical devices and host mucosal surfaces. However, the extracellular stimuli affecting these interactions are poorly understood. Based on our previous observations, we hypothesized that mucin, a glycoprotein secreted by lung epithelial cells, particularly during respiratory infections, significantly alters A. baumannii's physiology and its interaction with the surrounding environment. Biofilm, virulence and growth assays showed that mucin enhances the interaction of A. baumannii ATCC 19606T with abiotic and biotic surfaces and its cytolytic activity against epithelial cells while serving as a nutrient source. The global effect of mucin on the physiology and virulence of this pathogen is supported by RNA-Seq data showing that its presence in a low nutrient medium results in the differential transcription of 427 predicted protein-coding genes. The reduced expression of ion acquisition genes and the increased transcription of genes coding for energy production together with the detection of mucin degradation indicate that this host glycoprotein is a nutrient source. The increased expression of genes coding for adherence and biofilm biogenesis on abiotic and biotic surfaces, the degradation of phenylacetic acid and the production of an active type VI secretion system further supports the role mucin plays in virulence. Taken together, our observations indicate that A. baumannii recognizes mucin as an environmental signal, which triggers a response cascade that allows this pathogen to acquire critical nutrients and promotes host-pathogen interactions that play a role in the pathogenesis of bacterial infections.

Genetic and functional studies showed that some components of the Acinetobacter baumannii ATCC 17978 A1S_0112-A1S_0119 gene cluster are critical for biofilm biogenesis and surface motility. Recently, our group has shown that the A1S_0114 gene was involved in biofilm formation, a process related with pathogenesis. Confirming our previous results, microscopy images revealed that the ATCC 17978 10114 derivative lacking this gene was unable to form a mature biofilm structure. Therefore, other bacterial phenotypes were analyzed to determine the role of this gene in the pathogenicity of A. baumannii ATCC 17978. The interaction of the ATCC 17978 parental strain and the 10114 mutant with A549 human alveolar epithelial cells was quantified revealing that the A1S_0114 gene was necessary for proper attachment to A549 cells. This dependency correlates with the negative effect of the A1S_0114 deletion on the expression of genes coding for surface proteins and pili-assembly systems, which are known to play a role in adhesion. Three different experimental animal models, including vertebrate and invertebrate hosts, confirmed the role of the A1S_0114 gene in virulence. All of the experimental infection assays indicated that the virulence of the ATCC 17978 was significantly reduced when this gene was inactivated. Finally, we discovered that the A1S_0114 gene was involved in the production of a small lipopeptide-like compound herein referred to as acinetin 505 (Ac-505). Ac-505 was isolated from ATCC 17978 spent media and its chemical structure was interpreted by mass spectrometry. Overall, our observations provide novel information on the role of the A1S_0114 gene in A. baumannii’s pathobiology and lay the foundation for future work to determine the mechanisms by which Ac-505, or possibly an Ac-505 precursor, could execute critical functions as a secondary metabolite SI

Multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) strains of Acinetobacter baumannii have frequently been characterized. The ability of A. baumannii to develop resistance to antibiotics is a key reason this organism has been difficult to study using genetic and molecular biology approaches. Here we report selectable markers that are not only useful but necessary for the selection of drug-resistant transformants in the setting of drug-resistant backgrounds. Use of these selectable markers can be applied to a variety of genetic and molecular techniques such as mutagenesis and transformation. These selectable markers will help promote genetic and molecular biology studies of otherwise onerous drug-resistant strains, while avoiding the generation of pathogenic organisms that are resistant to clinically relevant antibiotics., Acinetobacter baumannii is one of the most antibiotic-resistant pathogens in clinical medicine, and extensively drug-resistant (XDR) strains are commonly isolated from infected patients. Such XDR strains are already resistant to traditional selectable genetic markers, limiting the ability to conduct pathogenesis research by genetic disruption. Optimization of selectable markers is therefore critical for the advancement of fundamental molecular biology techniques to use in these strains. We screened 23 drugs that constitute a broad array of antibiotics spanning multiple drug classes against HUMC1, a highly virulent and XDR A. baumannii clinical blood and lung isolate. HUMC1 is resistant to all clinically useful antibiotics that are reported by the clinical microbiology laboratory, except for colistin. Ethical concerns about intentionally establishing pan-resistance, including to the last-line agent, colistin, in a clinical isolate made identification of other markers desirable. We screened additional antibiotics that are in clinical use and those that are useful only in a lab setting to identify selectable markers that were effective at selecting for transformants in vitro. We show that supraphysiological levels of tetracycline can overcome innate drug resistance displayed by this XDR strain. Last, we demonstrate that transformation of the tetA (tetracycline resistance) and Sh ble (zeocin resistance), but not pac (puromycin resistance), resistance cassettes allow for selection of drug-resistant transformants. These results make the genetic manipulation of XDR A. baumannii strains easily achieved. IMPORTANCE Multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) strains of Acinetobacter baumannii have frequently been characterized. The ability of A. baumannii to develop resistance to antibiotics is a key reason this organism has been difficult to study using genetic and molecular biology approaches. Here we report selectable markers that are not only useful but necessary for the selection of drug-resistant transformants in the setting of drug-resistant backgrounds. Use of these selectable markers can be applied to a variety of genetic and molecular techniques such as mutagenesis and transformation. These selectable markers will help promote genetic and molecular biology studies of otherwise onerous drug-resistant strains, while avoiding the generation of pathogenic organisms that are resistant to clinically relevant antibiotics.

Fil: Mussi, Maria Alejandra. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Rosario. Centro de Estudios Fotosinteticos y Bioquimicos (i); Argentina

The Acinetobacter baumannii BlsA photoreceptor has an N-terminal (NT) BLUF domain and a C-terminal (CT) amino acid sequence with no significant homology to characterized bacterial proteins. In this study, we tested the biological role of specific residues located in these BlsA regions. Site-directed mutagenesis, surface motility assays at 24°C and protein overexpression showed that residues Y7, Q51 and W92 are essential for not only light-regulated motility, but also BlsA’s solubility when overexpressed in a heterologous host. In contrast, residues A29 and F32, the latter representing a difference when compared with other BLUF-containing photoreceptors, do not play a major role in BlsA’s biological functions. Analysis of the CT region showed that the deletion of the last five BlsA residues has no significant effect on the protein’s light-sensing and motility regulatory functions, but the deletion of the last 14 residues as well as K144E and K145E substitutions significantly alter light-regulated motility responses. In contrast to the NT mutants, these CT derivatives were overexpressed and purified to homogeneity to demonstrate that although these mutations do not significantly affect flavin binding and photocycling, they do affect BlsA’s photodynamic properties. Notably, these mutations map within a potential fifth α-helical component that could play a role in predicted interactions between regulatory partners and BlsA, which could function as a monomer according to gel filtration data. All these observations indicate that although BlsA shares common structural and functional properties with unrelated photoreceptors, it also exhibits unique features that make it a distinct BLUF photoreceptor.