Identification of Potential Virulence Factors in the Model Strain Acinetobacter baumannii A118.

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.

Isolation and Characterization of the Acinetobactin and Baumannoferrin Siderophores Produced by Acinetobacter baumannii.

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.

Antimicrobial Activity of Gallium Protoporphyrin IX against Acinetobacter baumannii Strains Displaying Different Antibiotic Resistance Phenotypes.

A paucity of effective, currently available antibiotics and a lull in antibiotic development pose significant challenges for treatment of patients with multidrug-resistant (MDR) Acinetobacter baumannii infections. Thus, novel therapeutic strategies must be evaluated to meet the demands of treatment of these often life-threatening infections. Accordingly, we examined the antibiotic activity of gallium protoporphyrin IX (Ga-PPIX) against a collection of A. baumannii strains, including nonmilitary and military strains and strains representing different clonal lineages and isolates classified as susceptible or MDR. Susceptibility testing demonstrated that Ga-PPIX inhibits the growth of all tested strains when cultured in cation-adjusted Mueller-Hinton broth, with a MIC of 20 µg/ml. This concentration significantly reduced bacterial viability, while 40 µg/ml killed all cells of the A. baumannii ATCC 19606(T) and ACICU MDR isolate after 24-h incubation. Recovery of ATCC 19606(T) and ACICU strains from infected A549 human alveolar epithelial monolayers was also decreased when the medium was supplemented with Ga-PPIX, particularly at a 40-µg/ml concentration. Similarly, the coinjection of bacteria with Ga-PPIX increased the survival of Galleria mellonella larvae infected with ATCC 19606(T) or ACICU. Ga-PPIX was cytotoxic only when monolayers or larvae were exposed to concentrations 16-fold and 1,250-fold higher than those showing antibacterial activity, respectively. These results indicate that Ga-PPIX could be a viable therapeutic option for treatment of recalcitrant A. baumannii infections regardless of the resistance phenotype, clone lineage, time and site of isolation of strains causing these infections and their iron uptake phenotypes or the iron content of the media.

Discovery and Characterization of New Hydroxamate Siderophores, Baumannoferrin A and B, produced by Acinetobacter baumannii.

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.

Molecular mechanisms of sulbactam antibacterial activity and resistance determinants in Acinetobacter baumannii.

Sulbactam is a class A ß-lactamase inhibitor with intrinsic whole-cell activity against certain bacterial species, including Acinetobacter baumannii. The clinical use of sulbactam for A. baumannii infections is of interest due to increasing multidrug resistance in this pathogen. However, the molecular drivers of its antibacterial activity and resistance determinants have yet to be precisely defined. Here we show that the antibacterial activities of sulbactam vary widely across contemporary A. baumannii clinical isolates and are mediated through inhibition of the penicillin-binding proteins (PBPs) PBP1 and PBP3, with very low frequency of resistance; the rare pbp3 mutants with high levels of resistance to sulbactam are attenuated in fitness. These results support further investigation of the potential clinical utility of sulbactam.

Role of the carboxy terminus of SecA in iron acquisition, protein translocation, and virulence of the bacterial pathogen Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative opportunistic nosocomial pathogen that causes pneumonia and soft tissue and systemic infections. Screening of a transposon insertion library of A. baumannii ATCC 19606T resulted in the identification of the 2010 derivative, which, although capable of growing well in iron-rich media, failed to prosper under iron chelation. Genetic, molecular, and functional assays showed that 2010's iron utilization-deficient phenotype is due to an insertion within the 3' end of secA, which results in the production of a C-terminally truncated derivative of SecA. SecA plays a critical role in protein translocation through the SecYEG membrane channel. Accordingly, the secA mutation resulted in undetectable amounts of the ferric acinetobactin outer membrane receptor protein BauA while not affecting the production of other acinetobactin membrane protein transport components, such as BauB and BauE, or the secretion of acinetobactin by 2010 cells cultured in the presence of subinhibitory concentrations of the synthetic iron chelator 2,2'-dipyridyl. Outer membrane proteins involved in nutrient transport, adherence, and biofilm formation were also reduced in 2010. The SecA truncation also increased production of 30 different proteins, including proteins involved in adaptation/tolerance responses. Although some of these protein changes could negatively affect the pathobiology of the 2010 derivative, its virulence defect is mainly due to its inability to acquire iron via the acinetobactin-mediated system. These results together indicate that although the C terminus of the A. baumannii ATCC 19606T SecA is not essential for viability, it plays a critical role in the production and translocation of different proteins and virulence.

AB5075, a Highly Virulent Isolate of Acinetobacter baumannii, as a Model Strain for the Evaluation of Pathogenesis and Antimicrobial Treatments.

UNLABELLED: Acinetobacter baumannii is recognized as an emerging bacterial pathogen because of traits such as prolonged survival in a desiccated state, effective nosocomial transmission, and an inherent ability to acquire antibiotic resistance genes. A pressing need in the field of A. baumannii research is a suitable model strain that is representative of current clinical isolates, is highly virulent in established animal models, and can be genetically manipulated. To identify a suitable strain, a genetically diverse set of recent U.S. military clinical isolates was assessed. Pulsed-field gel electrophoresis and multiplex PCR determined the genetic diversity of 33 A. baumannii isolates. Subsequently, five representative isolates were tested in murine pulmonary and Galleria mellonella models of infection. Infections with one strain, AB5075, were considerably more severe in both animal models than those with other isolates, as there was a significant decrease in survival rates. AB5075 also caused osteomyelitis in a rat open fracture model, while another isolate did not. Additionally, a Tn5 transposon library was successfully generated in AB5075, and the insertion of exogenous genes into the AB5075 chromosome via Tn7 was completed, suggesting that this isolate may be genetically amenable for research purposes. Finally, proof-of-concept experiments with the antibiotic rifampin showed that this strain can be used in animal models to assess therapies under numerous parameters, including survival rates and lung bacterial burden. We propose that AB5075 can serve as a model strain for A. baumannii pathogenesis due to its relatively recent isolation, multidrug resistance, reproducible virulence in animal models, and genetic tractability. IMPORTANCE: The incidence of A. baumannii infections has increased over the last decade, and unfortunately, so has antibiotic resistance in this bacterial species. A. baumannii is now responsible for more than 10% of all hospital-acquired infections in the United States and has a >50% mortality rate in patients with sepsis and pneumonia. Most research on the pathogenicity of A. baumannii focused on isolates that are not truly representative of current multidrug-resistant strains isolated from patients. After screening of a panel of isolates in different in vitro and in vivo assays, the strain AB5075 was selected as more suitable for research because of its antibiotic resistance profile and increased virulence in animal models. Moreover, AB5075 is susceptible to tetracycline and hygromycin, which makes it amenable to genetic manipulation. Taken together, these traits make AB5075 a good candidate for use in studying virulence and pathogenicity of this species and testing novel antimicrobials.

The Acinetobacter baumannii entA gene located outside the acinetobactin cluster is critical for siderophore production, iron acquisition and virulence.

Acinetobacter baumannii causes severe infections in compromised patients, who present an iron-limited environment that controls bacterial growth. This pathogen has responded to this restriction by expressing high-affinity iron acquisition systems including that mediated by the siderophore acinetobactin. Gene cloning, functional assays and biochemical tests showed that the A. baumannii genome contains a single functional copy of an entA ortholog. This gene, which is essential for the biosynthesis of the acinetobactin precursor 2,3-dihydroxybenzoic acid (DHBA), locates outside of the acinetobactin gene cluster, which otherwise harbors all genes needed for acinetobactin biosynthesis, export and transport. In silico analyses and genetic complementation tests showed that entA locates next to an entB ortholog, which codes for a putative protein that contains the isochorismatase lyase domain, which is needed for DHBA biosynthesis from isochorismic acid, but lacks the aryl carrier protein domain, which is needed for tethering activated DHBA and completion of siderophore biosynthesis. Thus, basF, which locates within the acinetobactin gene cluster, is the only fully functional entB ortholog present in ATCC 19606(T). The differences in amino acid length and sequences between these two EntB orthologs and the differences in the genetic context within which the entA and entB genes are found in different A. baumannii isolates indicate that they were acquired from different sources by horizontal transfer. Interestingly, the AYE strain proved to be a natural entA mutant capable of acquiring iron via an uncharacterized siderophore-mediated system, an observation that underlines the ability of different A. baumannii isolates to acquire iron using different systems. Finally, experimental infections using in vivo and ex vivo models demonstrate the role of DHBA and acinetobactin intermediates in the virulence of the ATCC 19606(T) cells, although to a lesser extent when compared to the responses obtained with bacteria producing and using fully matured acinetobactin to acquire iron.

Stress response and virulence functions of the Acinetobacter baumannii NfuA Fe-S scaffold protein.

To successfully establish an infection, Acinetobacter baumannii must overcome the iron starvation and oxidative stress imposed by the human host. Although previous studies have shown that ATCC 19606(T) cells acquire iron via the acinetobactin-mediated siderophore system, little is known about intracellular iron metabolism and its relation to oxidative stress in this pathogen. Screening of an insertion library resulted in the isolation of the ATCC 19606(T) derivative 1644, which was unable to grow in iron-chelated media. Rescue cloning and DNA sequencing showed that the insertion inactivated a gene coding for an NfuA Fe-S cluster protein ortholog, without any effect on the expression of the acinetobactin system. The nfuA mutant was also more sensitive to hydrogen peroxide and cumene hydroperoxide than the parental strain. The iron chelation- and oxidative-stress-deficient responses of this mutant were corrected when complemented with either the ATCC 19606(T) parental allele or the Escherichia coli MG1655 nfuA ortholog. Furthermore, electron paramagnetic resonance (EPR) and inductively coupled plasma-atomic emission spectroscopy (ICP-AES) analyses showed that the ATCC 19606(T) NfuA ortholog has iron-binding properties compatible with the formation of [Fe-S] cluster protein. Ex vivo and in vivo assays using human epithelial cells and Galleria mellonella, respectively, showed that NfuA is critical for bacterial growth independent of their capacity to acquire iron or the presence of excess of free iron. Taken together, these observations indicate that the A. baumannii NfuA ortholog plays a role in intracellular iron utilization and protection from oxidative-stress responses that this pathogen could encounter during the infection of the human host.

Iron acquisition functions expressed by the human pathogen Acinetobacter baumannii.

Acinetobacter baumannii is a gram-negative bacterium that causes serious infections in compromised patients. More recently, it has emerged as the causative agent of severe infections in military personnel wounded in Iraq and Afghanistan. This pathogen grows under a wide range of conditions including iron-limiting conditions imposed by natural and synthetic iron chelators. Initial studies using the type strain 19606 showed that the iron proficiency of this pathogen depends on the expression of the acinetobactin-mediated iron acquisition system. More recently, we have observed that hemin but not human hemoglobin serves as an iron source when 19606 isogenic derivatives affected in acinetobactin transport and biosynthesis were cultured under iron-limiting conditions. This finding is in agreement with the observation that the genome of the strain 17978 has a gene cluster coding for putative hemin-acquisition functions, which include genes coding for putative hemin utilization functions and a TonBExbBD energy transducing system. This system restored enterobactin biosynthesis in an E. coli ExbBD deficient strain but not when introduced into a TonB mutant. PCR and Southern blot analyses showed that this hemin-utilization gene cluster is also present in the 19606 strain. Analysis of the 17978 genome also showed that this strain harbors genes required for acinetobactin synthesis and transport as well as a gene cluster that could code for additional iron acquisition functions. This hypothesis is in agreement with the fact that the inactivation of the basD acinetobactin biosynthetic gene did not affect the growth of A. baumannii 17978 cells under iron-chelated conditions. Interestingly, this second iron uptake gene cluster is flanked by perfect inverted repeats and includes transposase genes that are expressed transcriptionally. Also interesting is the observation that this additional cluster could not be detected in the type strain 19606, an observation that suggests some significant differences in the iron uptake capacity between these two A. baumannii strains. Transposome mutagenesis of the strain 19606 resulted in the isolation of a derivative unable to grow under iron-chelated conditions. Gene mapping and protein analysis together with complementation assays showed that a protein related to SecA, which is a component of the Sec protein secretion system in a wide range of bacteria, is needed at least for the production of the BauA acinetobactin outer membrane receptor. Furthermore, this derivative was unable to use hemin as an iron source under limiting conditions. Taken together, these results indicate that A. baumannii expresses siderophore-mediated and hemin acquisition functions, although different isolates differ in their iron acquisition capacity. Unexpectedly, the ability of this pathogen to acquire iron depends on the expression of a SecA protein secretion function, which has not been associated with iron acquisition in bacteria.