Janet M. Beanan

The K1 Capsular Polysaccharide of Acinetobacter baumannii Strain 307-0294 Is a Major Virulence Factor

ABSTRACT Acinetobacter baumannii is a pathogen of increasing medical importance with a propensity to be multidrug resistant, thereby making treatment challenging. Little is known of virulence traits in A. baumannii. To identify virulence factors and potential drug targets, random transposon (Tn) mutants derived from the A. baumannii strain AB307-0294 were screened to identify genes essential for growth in human ascites fluid in vitro, an inflammatory exudative fluid. These studies led to the identification of two genes that were predicted to be required for capsule polymerization and assembly. The first, ptk, encodes a putative protein tyrosine kinase (PTK), and the second, epsA, encodes a putative polysaccharide export outer membrane protein (EpsA). Monoclonal antibodies used in flow cytometric and Western analyses confirmed that these genes are required for a capsule-positive phenotype. A capsule-positive phenotype significantly optimized growth in human ascites fluid, survival in human serum, and survival in a rat soft tissue infection model. Importantly, the clearance of the capsule-minus mutants AB307.30 (ptk mutant, capsule minus) and AB307.45 (epsA mutant, capsule minus) was complete and durable. These data demonstrated that the K1 capsule from AB307-0294 was an important protectin. Further, these data suggested that conserved proteins, which contribute to the capsule-positive phenotype, are potential antivirulence drug targets. Therefore, the results from this study have important biologic and translational implications and, to the best of our knowledge, are the first to address the role of capsule in the pathogenesis of A. baumannii infection.

IroN Functions as a Siderophore Receptor and Is a Urovirulence Factor in an Extraintestinal Pathogenic Isolate of Escherichia coli

ABSTRACT IroN was recently identified in the extracellular pathogenic Escherichia coli strain CP9. In this study experimental evidence demonstrating that IroN mediates utilization of the siderophore enterobactin was obtained, thereby establishing IroN as a catecholate siderophore receptor. In a mouse model of ascending urinary tract infection the presence of iroN contributed significantly to CP9s ability to colonize the mouse bladder, kidneys, and urine, evidence that IroN is a urovirulence factor. However, growth in human urine ex vivo and adherence to uroepithelial cells in vitro were equivalent for an isogenic mutant deficient in IroN (CP82) and its wild-type parent (CP9). Taken together, these findings establish that IroN is a siderophore receptor and a urovirulence factor. However, uncertainty exists as to the mechanism(s) via which IroN contributes to urovirulence.

The Siderophore Receptor IroN of Extraintestinal Pathogenic Escherichia coli Is a Potential Vaccine Candidate

ABSTRACT It would be medically and economically desirable to prevent the millions of annual extraintestinal infections and the thousands of associated deaths due to Escherichia coli. Outer membrane proteins are potential vaccine candidates for the prevention of these infections. This study tested the hypotheses that the siderophore receptor IroN is antigenic and that an IroN-specific antibody response confers protection in vivo. Subcutaneous immunization with denatured IroN resulted in a significant IroN immunoglobulin G (IgG)-specific response in serum (P < 0.0001) but not a systemic or mucosal IroN-specific IgA response. In a mouse model of ascending urinary tract infection, subcutaneous immunization with denatured IroN conferred significant protection against renal (P = 0.0135 and 0.0095 in two independent experiments), but not bladder, infection. These data, together with the previously demonstrated role of IroN in virulence, its expression in human biologic fluids, and its prevalence among extraintestinal pathogenic E. coli strains, support further studies on the role of IroN as a vaccine candidate.

Clinical and phenotypic differences between classic and hypervirulent Klebsiella pneumonia: an emerging and under-recognized pathogenic variant

The purpose of this study was to increase awareness, gain insight into acquisition, and assess the virulence of the hypervirulent (hypermucoviscous) clinical variant (hvKP) that is entrenched in the Pacific Rim but emerging in Western countries. A case of community-acquired liver abscess with metastatic spread to the spleen is described. Comparative in vitro and in vivo virulence studies on this isolate (hvKP1) and four randomly chosen blood isolates of “classic” K. pneumonia strains (cKP1-4) were performed. Cases of hvKP infection are occurring in Western countries and are under-recognized. A hypermucoviscous phenotype is a surrogate laboratory marker for this variant. The propensity of hvKP strains for metastatic spread in non-compromised hosts is both a defining and unusual trait. The mode of acquisition in the described case was unclear but potential means are discussed. hvKP1 was more resistant to complement and neutrophil-mediated bactericidal activity and was more virulent in a rat subcutaneous abscess model than cKP1-4. Recognition of the hypermucoviscous phenotype, defined by a positive “string-test”, will alert the microbiologist or clinician that the infecting strain may be a hvKP, which is hypervirulent compared to cKP. This will improve our understanding of the epidemiology and clinical spectrum of infection, which may be more extensive than appreciated.

Penicillin-binding protein 7/8 contributes to the survival of Acinetobacter baumannii in vitro and in vivo

BACKGROUND Acinetobacter baumannii is a bacterial pathogen of increasing medical importance. Little is known about genes important for its survival in vivo. METHODS AND RESULTS Screening of random transposon mutants of the model pathogen AB307-0294 identified the mutant AB307.27. AB307.27 contained its transposon insertion in pbpG, which encodes the putative low-molecular-mass penicillin-binding protein 7/8 (PBP-7/8). AB307.27 was significantly killed in ascites (P<.001), but its growth in Luria-Bertani broth was similar to that of its parent, AB307-0294 (P=.13). The survival of AB307.27 was significantly decreased in a rat soft-tissue infection model (P<.001) and a rat pneumonia model (P=.002), compared with AB307-0294. AB307.27 was significantly killed in 90% human serum in vitro, compared with AB307-0294 (P<.001). Electron microscopy demonstrated more coccobacillary forms of AB307.27, compared with AB307-0294, suggesting a possible modulation in the peptidoglycan, which may affect susceptibility to host defense factors. CONCLUSIONS These findings demonstrate that PBP-7/8 contributes to the pathogenesis of A. baumannii. PBP-7/8 either directly or indirectly contributes to the resistance of AB307-0294 to complement-mediated bactericidal activity. An understanding of how PBP-7/8 contributes to serum resistance will lend insight into the role of this low-molecular-mass PBP whose function is poorly understood.

The K1 Capsular Polysaccharide from Acinetobacter baumannii Is a Potential Therapeutic Target via Passive Immunization

ABSTRACT The emergence of extremely resistant and panresistant Gram-negative bacilli, such as Acinetobacter baumannii, requires consideration of nonantimicrobial therapeutic approaches. The goal of this report was to evaluate the K1 capsular polysaccharide from A. baumannii as a passive immunization target. Its structure was determined by a combination of mass spectrometric and nuclear magnetic resonance (NMR) techniques. Molecular mimics that might raise the concern for autoimmune disease were not identified. Immunization of CD1 mice demonstrated that the K1 capsule is immunogenic. The monoclonal antibody (MAb) 13D6, which is directed against the K1 capsule from A. baumannii, was used to determine the seroprevalence of the K1 capsule in a collection of 100 A. baumannii strains. Thirteen percent of the A. baumannii isolates from this collection were seroreactive to MAb 13D6. Opsonization of K1-positive strains, but not K1-negative strains, with MAb 13D6 significantly increased neutrophil-mediated bactericidal activity in vitro (P < 0.05). Lastly, treatment with MAb 13D6 3 and 24 h after bacterial challenge in a rat soft tissue infection model resulted in a significant decrease in the growth/survival of a K1-positive strain compared to that of a K1-negative strain or to treatment with a vehicle control (P < 0.0001). These data support the proof of principle that the K1 capsule is a potential therapeutic target via passive immunization. Other serotypes require assessment, and pragmatic challenges exist, such as the need to serotype infecting strains and utilize serotype-specific therapy. Nonetheless, this approach may become an important therapeutic option with increasing antimicrobial resistance and a diminishing number of active antimicrobials.

Identification and Characterization of a Glycosyltransferase Involved in Acinetobacter baumannii Lipopolysaccharide Core Biosynthesis

ABSTRACT Although Acinetobacter baumannii has emerged as a significant cause of nosocomial infections worldwide, there have been few investigations describing the factors important for A. baumannii persistence and pathogenesis. This paper describes the first reported identification of a glycosyltransferase, LpsB, involved in lipopolysaccharide (LPS) biosynthesis in A. baumannii. Mutational, structural, and complementation analyses indicated that LpsB is a core oligosaccharide glycosyl transferase. Using a genetic approach, lpsB was compared with the lpsB homologues of several A. baumannii strains. These analyses indicated that LpsB is highly conserved among A. baumannii isolates. Furthermore, we developed a monoclonal antibody, monoclonal antibody 13C11, which reacts to an LPS core epitope expressed by approximately one-third of the A. baumannii clinical isolates evaluated to date. Previous studies describing the heterogeneity of A. baumannii LPS were limited primarily to structural analyses; therefore, studies evaluating the correlation between these surface glycolipids and pathogenesis were warranted. Our data from an evaluation of LpsB mutant 307::TN17, which expresses a deeply truncated LPS glycoform consisting of only two 3-deoxy-d-manno-octulosonic acid residues and lipid A, suggest that A. baumannii LPS is important for resistance to normal human serum and confers a competitive advantage for survival in vivo. These results have important implications for the role of LPS in A. baumannii infections.

In vivo and in vitro activity of the siderophore monosulfactam BAL30072 against Acinetobacter baumannii

OBJECTIVES New antibiotics that are active against multidrug-resistant (MDR) Acinetobacter baumannii are urgently needed. BAL30072, a siderophore monosulfactam antibiotic that rapidly penetrates the outer membrane of A. baumannii and has potent activity against most isolates, including those harbouring AmpC β-lactamases and metallo- (class B) or OXA- (class D) carbapenemases, is being developed to meet that need. METHODS We assessed the in vitro activity of BAL30072, meropenem and the combination of BAL30072 and meropenem (2:1 and 1:1 ratios) by MIC and time-kill studies. Proof-of-principle in vivo efficacy was determined using a rat soft-tissue infection model. Five diverse strains with defined phenotypic and genetic profiles were tested (AB307-0294, AB8407, AB1697, AB3340 and AB0057). RESULTS In microdilution assays, combining BAL30072 with meropenem lowered meropenem MICs 2-8-fold. In time-kill studies, the BAL30072 and meropenem combinations resulted in bactericidal concentrations 2-8-fold lower than those of meropenem or BAL30072 alone. In the rat model, BAL30072 was active against four of five strains (AB307-0294, AB8407, AB1697 and AB3340), including meropenem-susceptible and -non-susceptible strains. AB0057 was the only strain resistant to BAL30072 in vivo and in vitro (MIC >64 mg/L). Meropenem was active in vivo against two of the five strains tested (AB307-0294 and AB3340). Both BAL30072 and BAL30072 with meropenem were equally effective in vivo. CONCLUSIONS These data support the continued evaluation of BAL30072 for use in the treatment of infections caused by MDR A. baumannii.

Hypervirulent K. Pneumoniae Secretes More and More Active Iron-Acquisition Molecules than “Classical” K. Pneumoniae Thereby Enhancing its Virulence

Background A new hypervirulent (hypermucoviscous) clinical variant of Klebsiella pneumoniae (hvKP) has emerged over the last decade. Our goal is to identify new mechanisms, which increase the virulence hvKP compared to “classic” K. pneumoniae (cKP). Methodology/Principal Findings Various growth assays were performed in human ascites, human serum, and laboratory medium with the hvKP strain hvKP1 (wt), randomly chosen blood isolates of cKP strains (cKP1-4), and mutant constructs deficient in the secretion of selected compounds. An in vivo mouse model that mimics infection due to hvKP and a quantitative siderophore assay were also used. It was established that a molecule(s)/factor(s) was secreted by hvKP1 significantly enhanced its growth and/or survival in human ascites. This molecule(s)/factor(s) also increased the growth and/or survival of hvKP1 in serum ex vivo and in an in vivo mouse model that measures metastatic spread after subcutaneous challenge, thereby further establishing biologic significance. Although features such as a size of <3kD, heat stability, and growth characteristics in ascites suggested this molecule(s) was a quorum-sensing compound, data presented demonstrates that this molecule(s)/factor(s) is involved in iron uptake and is likely a siderophore(s) or another iron-acquisition molecule. Although it is known that iron acquisition is critical for virulence, a novel aspect of this observation is that hvKP1 produces quantitatively more siderophores that appear to be biologically more active (increased affinity for iron or more resistant to host factors) than those produced by cKP strains. Conclusions/Significance The data presented delineates a new mechanism by which hvKP increases its pathogenic potential compared to cKP strains. This paradigm may be broadly applicable to other extraintestinal gram-negative bacilli.

Rat Pneumonia and Soft-Tissue Infection Models for the Study of Acinetobacter baumannii Biology

ABSTRACT Acinetobacter baumannii is a bacterial pathogen of increasing medical importance. Little is known about its mechanisms of pathogenesis, and safe reliable agents with predictable activity against A. baumannii are presently nonexistent. The availability of relevant animal infection models will facilitate the study of Acinetobacter biology. In this report we tested the hypothesis that the rat pneumonia and soft-tissue infection models that our laboratory had previously used for studies of extraintestinal pathogenic Escherichia coli were clinically relevant for A. baumannii. Advantages of these models over previously described models were that the animals were not rendered neutropenic and they did not receive porcine mucin with bacterial challenge. Using the A. baumannii model pathogen 307-0294 as the challenge pathogen, the pneumonia model demonstrated all of the features of infection that are critical for a clinically relevant model: namely, bacterial growth/clearance, an ensuing host inflammatory response, acute lung injury, and, following progressive bacterial proliferation, death due to respiratory failure. We were also able to demonstrate growth of 307-0294 in the soft-tissue infection model. Next we tested the hypothesis that the soft-tissue infection model could be used to discriminate between the inherent differences in virulence of various A. baumannii clinical isolates. The ability of A. baumannii to grow and/or be cleared in this model was dependent on the challenge strain. We also hypothesized that complement is an important host factor in protecting against A. baumannii infection in vivo. In support of this hypothesis was the observation that the serum sensitivity of various A. baumannii clinical isolates in vitro roughly paralleled their growth/clearance in the soft-tissue infection model in vivo. Lastly we hypothesized that the soft-tissue infection model would serve as an efficient screening mechanism for identifying gene essentiality for drug discovery. Random mutants of 307-0294 were initially screened for lack of growth in human ascites in vitro. Selected mutants were subsequently used for challenge in the soft-tissue infection model to determine if the disrupted gene was essential for growth in vivo. Using this approach, we have been able to successfully identify a number of genes essential for the growth of 307-0294 in vivo. In summary, these models are clinically relevant and can be used to study the innate virulence of various Acinetobacter clinical isolates and to assess potential virulence factors, vaccine candidates, and drug targets in vivo and can be used for pharmacokinetic and chemotherapeutic investigations.