Methodological approach: using a within-subjects design in the marble burying assay.

In 2016, the National Institutes of Health mandated the use of both male and female mice in funded research. The use of both sexes is an important variable to consider; however, it comes with negative consequences such as increased animal expenses. One way to combat these negatives is to explore the option of using a within-subjects design (repeated measures) in behavioral assays that historically use a between-subjects design. Our study aimed to determine if a within-subjects design can be utilized in the marble burying assay. The marble burying assay is used as a tool for screening putative anxiolytic compounds as the assay is thought to measure obsessive-compulsive disorder- or anxiety-like behaviors. First, we compared the effects of sex and digging medium (corn cob or Sani Chip) on the number of marbles buried using CD-1 mice. Second, we determined if mice would continue to bury marbles after repeated exposures to the test arena. Lastly, we tested three positive controls (buspirone, ketamine, and fluoxetine). We found that mice buried significantly more marbles within Sani Chip digging medium, and no sex differences were observed. Next, the number of marbles buried and locomotor activity remained consistent across four test sessions. The positive controls buspirone (3.2-10 mg/kg) ketamine (32 mg/kg), and fluoxetine (10 mg/kg) decreased the number of marbles buried using the within-subjects design. These data suggest that a within-subjects design is optimal for the marble burying assay as it will reduce the number of animals and increase statistical power.

Repurposed dihydroorotate dehydrogenase inhibitors with efficacy against drug-resistant Acinetobacter baumannii.

New antimicrobials are needed for the treatment of extensively drug-resistant Acinetobacter baumannii. The de novo pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) is a validated drug target for malaria and human autoimmune diseases. We provide genetic evidence that A. baumannii DHODH (AbDHODH) is essential for bacterial survival in rodent infection models. We chemically validate the target by repurposing a unique library of ~450 triazolopyrimidine/imidazopyrimidine analogs developed for our malaria DHODH program to identify 21 compounds with submicromolar activity on AbDHODH. The most potent (DSM186, DHODH IC50 28 nM) had a minimal inhibitory concentration of ≤1 µg/ml against geographically diverse A. baumannii strains, including meropenem-resistant isolates. A structurally related analog (DSM161) with a long in vivo half-life conferred significant protection in the neutropenic mouse thigh infection model. Encouragingly, the development of resistance to these compounds was not identified in vitro or in vivo. Lastly, the X-ray structure of AbDHODH bound to DSM186 was solved to 1.4 Å resolution. These data support the potential of AbDHODH as a drug target for the development of antimicrobials for the treatment of A. baumannii and potentially other high-risk bacterial infections.

Ionic strength and polyelectrolyte molecular weight effects on floc formation and growth in Taylor-Couette flows.

Polyelectrolyte-driven flocculation of suspended particulate in solution is an important process in a variety of industrial processes such as drinking water treatment and composite material synthesis. Flocculation depends on a wide variety of physicochemical and hydrodynamic properties, which affect floc size, growth rate, and floc morphology. Floc formation and growth behavior is explored here using two different molecular weights of a cationic polyacrylamide flocculant and anisotropic Na-bentonite clay particles under a variety of solution ionic strengths. A Taylor-Couette cell with radial injection capabilities was used to study the effects of solution ionic strength and polyelectrolyte molecular weight on floc size, growth rate, and floc morphology during the flocculation process with a constant global velocity gradient. The floc size generally decreased with increasing ionic strength whereas the floc growth rate initially increased then decreased. This likely occurred due to charge screening effects, where increased bentonite aggregate size and a less expanded polyelectrolyte conformation at higher ionic strengths results in a decreased ability for the polyelectrolyte to bridge multiple bentonite aggregates. The densification of bentonite aggregates at higher ionic strengths resulted in floc morphologies that were more resistant to shear-induced breakage. With the exceptions of optimal dose concentration and dispersion coefficients, there were no clear differences in the floc growth rate behaviors for the two molecular weights studied. This work contributes to an improved understanding of the physicochemical complexities of polyelectrolyte-driven flocculation that can inform dosing requirements for more efficient industrial operations.

Antibody Dependent Enhancement of Acinetobacter baumannii Infection in a Mouse Pneumonia Model.

Acinetobacter baumannii has become a pathogen of increasing medical importance because of the emergence of multidrug-resistant strains and the high rate of mortality of infected patients. Promising animal study results have been reported recently with active and passive immunization against A. baumannii virulence factors. In the present study, a monoclonal IgG3 antibody, 8E3, was developed with specificity for the K2 capsular polysaccharide of A. baumannii, and its therapeutic potential was assessed. 8E3 enhanced macrophage-mediated bactericidal activity against the A. baumannii clinical strain AB899. However, 8E3 treatment (passive immunization) of AB899-infected mice led to a substantial increase in mortality and to substantial increases in bacterial load in blood, lung, and in splenic samples. In vitro investigations showed a large binding capacity in the supernatant of bacterial cultures, suggesting that shed capsule components act as a binding sink for 8E3. Investigations of 8E3 pharmacokinetics in mice demonstrated that unbound concentrations of the antibody dropped below detection limits within 24 hours after a 200 mg/kg dose. However, total concentrations of antibody declined slowly, with an apparent terminal half-life (t 1/2) of 6.7-8.0 days, suggesting that the vast majority of 8E3 in blood is bound (e.g., with soluble capsule components in blood). We hypothesize that high concentrations of 8E3-capsule immune complexes act to inhibit bacterial clearance in vivo. To the best of our knowledge, this is the first demonstration of antibody-dependent enhancement of A. baumannii infection, and these observations highlight the complexity of antibody-based therapy for A. baumannii infections.

Identification of Biomarkers for Differentiation of Hypervirulent Klebsiella pneumoniae from Classical K. pneumoniae.

A hypervirulent Klebsiella pneumoniae (hvKp) pathotype is undergoing global dissemination. In contrast to the usual health care-associated epidemiology of classical K. pneumoniae (cKp) infections, hvKp causes tissue-invasive infections in otherwise healthy individuals from the community, often involving multiple sites. An accurate test to identify hvKp strains is needed for improved patient care and epidemiologic studies. To fill this knowledge gap, clinical criteria or random blood isolates from North American and United Kingdom strain collections were used to assemble hvKp-rich (n = 85) and cKp-rich (n = 90) strain cohorts, respectively. The isolates were then assessed for multiple candidate biomarkers hypothesized to accurately differentiate the two cohorts. The genes peg-344, iroB, iucA, plasmid-borne rmpA gene ( prmpA), and prmpA2 all demonstrated >0.95 diagnostic accuracy for identifying strains in the hvKp-rich cohort. Next, to validate this epidemiological analysis, all strains were assessed experimentally in a murine sepsis model. peg-344, iroB, iucA, prmpA, and prmpA2 were all associated with a hazard ratio of >25 for severe illness or death, additionally supporting their utility for identifying hvKp strains. Quantitative siderophore production of ≥30 µg/ml also strongly predicted strains as members of the hvKp-rich cohort (accuracy, 0.96) and exhibited a hazard ratio of 31.7 for severe illness or death. The string test, a widely used marker for hvKp strains, performed less well, achieving an accuracy of only 0.90. Last, using the most accurate biomarkers to define hvKp, prevalence studies were performed on two Western strain collections. These data strongly support the utility of several laboratory markers for identifying hvKp strains with a high degree of accuracy.

The Capsular Polysaccharide of Acinetobacter baumannii Is an Obstacle for Therapeutic Passive Immunization Strategies.

Acinetobacter baumannii has become an important concern for human health due to rapid development and wide spread of antimicrobial-resistant strains and high mortality associated with the infection. Passive immunizations with antisera targeting outer membrane proteins (OMPs) have shown encouraging results in protecting mice from A. baumannii infection, but monoclonal anti-OMP antibodies have not been developed, and their potential therapeutic properties have not been explored. The goal of this report is to evaluate the antibacterial activity of monoclonal antibodies (MAbs) targeting outer membrane protein A (OmpA) of A. baumannii Five anti-OmpA MAbs were developed using hybridoma technology and showed strong binding to strain ATCC 19606. However, low antibody binding was observed when they were tested against six clinical isolates, which included extensively drug-resistant strains. In contrast, high binding to an isogenic K1 capsule-negative mutant (AB307.30) was shown, suggesting that capsular polysaccharide mediated the inhibition of MAb binding to OmpA. Anti-OmpA MAbs increased the macrophage-mediated bactericidal activity of AB307.30 but failed to increase phagocytic killing of capsule-positive strains. Capsular polysaccharide was also protective against complement-mediated bactericidal activity in human ascites in the presence and absence of opsonization. Lastly, passive immunization with anti-OmpA MAbs did not confer protection against challenge with AB307-0294, the encapsulated parent strain of AB307.30, in a mouse sepsis infection model. These results reveal the important role of capsule polysaccharide in shielding OmpA and thereby inhibiting anti-OmpA MAb binding to clinical isolates. This property of capsule hindered the therapeutic utility of anti-OmpA MAbs, and it may apply to other conserved epitopes in A. baumannii.

Metabolite Transporter PEG344 Is Required for Full Virulence of Hypervirulent Klebsiella pneumoniae Strain hvKP1 after Pulmonary but Not Subcutaneous Challenge.

Hypervirulent Klebsiella pneumoniae (hvKP) is an emerging pathotype that is capable of causing tissue-invasive and organ- and life-threatening infections in healthy individuals from the community. Knowledge on the virulence factors specific to hvKP is limited. In this report, we describe a new factor (PEG344) that increases the virulence of hvKP strain hvKP1. peg-344 is present on the hvKP1 virulence plasmid, is broadly prevalent among hvKP strains, and has increased RNA abundance when grown in human ascites. An isogenic derivative of hvKP1 (hvKP1Δpeg-344) was constructed and compared with its wild-type parent strain in in vitro, ex vivo, and infection model studies. Both survival and competition experiments with outbred CD1 mice demonstrated that PEG344 was required for full virulence after pulmonary challenge but, interestingly, not after subcutaneous challenge. In silico analysis suggested that PEG344 serves as an inner membrane transporter. Compared to hvKP1, a small but significant decrease in the growth/survival of hvKP1Δpeg-344 was observed in human ascites, but resistance to the bactericidal activity of complement was similar. These data suggested that PEG344 may transport an unidentified growth factor present in ascites. The data presented are important since they expand our limited knowledge base on virulence factors unique to hvKP, which is needed to lay the groundwork for translational approaches to prevent or treat these devastating infections.

The Response Regulator BfmR Is a Potential Drug Target for Acinetobacter baumannii.

Identification and validation is the first phase of target-based antimicrobial development. BfmR (RstA), a response regulator in a two-component signal transduction system (TCS) in Acinetobacter baumannii, is an intriguing potential antimicrobial target. A unique characteristic of BfmR is that its inhibition would have the dual benefit of significantly decreasing in vivo survival and increasing sensitivity to selected antimicrobials. Studies on the clinically relevant strain AB307-0294 have shown BfmR to be essential in vivo. Here, we demonstrate that this phenotype in strains AB307-0294 and AB908 is mediated, in part, by enabling growth in human ascites fluid and serum. Further, BfmR conferred resistance to complement-mediated bactericidal activity that was independent of capsular polysaccharide. Importantly, BfmR also increased resistance to the clinically important antimicrobials meropenem and colistin. BfmR was highly conserved among A. baumannii strains. The crystal structure of the receiver domain of BfmR was determined, lending insight into putative ligand binding sites. This enabled an in silico ligand binding analysis and a blind docking strategy to assess use as a potential druggable target. Predicted binding hot spots exist at the homodimer interface and the phosphorylation site. These data support pursuing the next step in the development process, which includes determining the degree of inhibition needed to impact growth/survival and the development a BfmR activity assay amenable to high-throughput screening for the identification of inhibitors. Such agents would represent a new class of antimicrobials active against A. baumannii which could be active against other Gram-negative bacilli that possess a TCS with shared homology. IMPORTANCE Increasing antibiotic resistance in bacteria, particularly Gram-negative bacilli, has significantly affected the ability of physicians to treat infections, with resultant increased morbidity, mortality, and health care costs. In fact, some strains of bacteria are resistant to all available antibiotics, such as Acinetobacter baumannii, which is the focus of this report. Therefore, the development of new antibiotics active against these resistant strains is urgently needed. In this study, BfmR is further validated as an intriguing target for a novel class of antibiotics. Successful inactivation of BfmR would confer the multiple benefits of a decreased ability of A. baumannii to survive in human body fluids, increased sensitivity to complement-mediated bactericidal activity and, importantly, increased sensitivity to other antibiotics. Structural studies support the potential for this "druggable" target, as they identify the potential for small-molecule binding at functionally relevant sites. Next-phase high-throughput screening studies utilizing BfmR are warranted.

Structure of shikimate kinase, an in vivo essential metabolic enzyme in the nosocomial pathogen Acinetobacter baumannii, in complex with shikimate.

Acinetobacter baumannii is an opportunistic Gram-negative pathogen that is an important cause of healthcare-associated infections exhibiting high mortality rates. Clinical isolates of multidrug-resistant (MDR) and extremely drug-resistant (XDR) A. baumannii strains are increasingly being observed. Compounding this concern is the dearth of new antibacterial agents in late-stage development that are effective against MDR and XDR A. baumannii. As part of an effort to address these concerns, two genes (aroA and aroC) of the shikimate pathway have previously been determined to be essential for the growth and survival of A. baumannii during host infection (i.e. to be essential in vivo). This study expands upon these results by demonstrating that the A. baumannii aroK gene, encoding shikimate kinase (SK), is also essential in vivo in a rat soft-tissue infection model. The crystal structure of A. baumannii SK in complex with the substrate shikimate and a sulfate ion that mimics the binding interactions expected for the ß-phosphate of ATP was then determined to 1.91 Å resolution and the enzyme kinetics were characterized. The flexible shikimate-binding domain and LID region are compared with the analogous regions in other SK crystal structures. The impact of structural differences and sequence divergence between SKs from pathogenic bacteria that may influence antibiotic-development efforts is discussed.

Aerobactin, but not yersiniabactin, salmochelin, or enterobactin, enables the growth/survival of hypervirulent (hypermucoviscous) Klebsiella pneumoniae ex vivo and in vivo.

The siderophore aerobactin is the dominant siderophore produced by hypervirulent Klebsiella pneumoniae (hvKP) and was previously shown to be a major virulence factor in systemic infection. However, strains of hvKP commonly produce the additional siderophores yersiniabactin, salmochelin, and enterobactin. The roles of these siderophores in hvKP infection have not been optimally defined. To that end, site-specific gene disruptions were created in hvKP1 (wild type), resulting in the generation of hvKP1ΔiucA (aerobactin deficient), hvKP1ΔiroB (salmochelin deficient), hvKP1ΔentB (enterobactin and salmochelin deficient), hvKP1Δirp2 (yersiniabactin deficient), and hvKP1ΔentBΔirp2 (enterobactin, salmochelin, and yersiniabactin deficient). The growth/survival of these constructs was compared to that of their wild-type parent hvKP1 ex vivo in human ascites fluid, human serum, and human urine and in vivo in mouse systemic infection and pulmonary challenge models. Interestingly, in contrast to aerobactin, the inability to produce enterobactin, salmochelin, or yersiniabactin individually or in combination did not decrease the ex vivo growth/survival in human ascites or serum or decrease virulence in the in vivo infection models. Surprisingly, none of the siderophores increased growth in human urine. In human ascites fluid supplemented with exogenous siderophores, siderophores increased the growth of hvKP1ΔiucA, with the relative activity being enterobactin > aerobactin > yersiniabactin > salmochelin, suggesting that the contribution of aerobactin to virulence is dependent on both innate biologic activity and quantity produced. Taken together, these data confirm and extend a role for aerobactin as a critical virulence factor for hvKP. Since it appears that aerobactin production is a defining trait of hvKP strains, this factor is a potential antivirulence target.

Aerobactin mediates virulence and accounts for increased siderophore production under iron-limiting conditions by hypervirulent (hypermucoviscous) Klebsiella pneumoniae.

Hypervirulent (hypermucoviscous) Klebsiella pneumoniae (hvKP) strains are an emerging variant of "classical" K. pneumoniae (cKP) that cause organ and life-threatening infection in healthy individuals. An understanding of hvKP-specific virulence mechanisms that enabled evolution from cKP is limited. Observations by our group and previously published molecular epidemiologic data led us to hypothesize that hvKP strains produced more siderophores than cKP strains and that this trait enhanced hvKP virulence. Quantitative analysis of 12 hvKP strains in iron-poor minimal medium or human ascites fluid showed a significant and distinguishing 6- to 10-fold increase in siderophore production compared to that for 14 cKP strains. Surprisingly, high-pressure liquid chromatography (HPLC)-mass spectrometry and characterization of the hvKP strains hvKP1, A1142, and A1365 and their isogenic aerobactin-deficient (ΔiucA) derivatives established that aerobactin accounted for the overwhelming majority of increased siderophore production and that this was not due to gene copy number. Further, aerobactin was the primary factor in conditioned medium that enhanced the growth/survival of hvKP1 in human ascites fluid. Importantly the ex vivo growth/survival of hvKP1 ΔiucA was significantly less than that of hvKP1 in human ascites fluid, and the survival of outbred CD1 mice challenged subcutaneously or intraperitoneally with hvKP1 was significantly less than that of mice challenged with hvKP1 ΔiucA. The lowest subcutaneous and intraperitoneal challenge inocula of 3 × 10(2) and 3.2 × 10(1) CFU, respectively, resulted in 100% mortality, demonstrating the virulence of hvKP1 and its ability to cause infection at a low dose. These data strongly support that aerobactin accounts for increased siderophore production in hvKP compared to cKP (a potential defining trait) and is an important virulence factor.

OxyR contributes to the virulence of a Clonal Group A Escherichia coli strain (O17:K+:H18) in animal models of urinary tract infection, subcutaneous infection, and systemic sepsis.

The oxidative stress response regulator OxyR was assessed as both a urinary and extra-urinary virulence factor in Escherichia coli strain UCB34 (O17:K+:H18), a representative of the emergent Clonal Group A (CGA). Compared to UCB34, the isogenic oxyR mutant exhibited increased H2O2 sensitivity, indistinguishable in vitro growth, and attenuated virulence in rodent models of urinary tract, subcutaneous infection, and systemic sepsis. Complemented mutants showed virulence levels comparable to parent strains in all models. These findings uniquely fulfill molecular Koch's postulates for a putative virulence factor of CGA, provide experimental evidence of an extra-urinary virulence promoting trait in CGA, and document a role for OxyR in local and systemic extra-urinary E. coli infections.

The K1 capsular polysaccharide from Acinetobacter baumannii is a potential therapeutic target via passive immunization.

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.

In vivo-validated essential genes identified in Acinetobacter baumannii by using human ascites overlap poorly with essential genes detected on laboratory media.

UNLABELLED: A critical feature of a potential antimicrobial target is the characteristic of being essential for growth and survival during host infection. For bacteria, genome-wide essentiality screens are usually performed on rich laboratory media. This study addressed whether genes detected in that manner were optimal for the identification of antimicrobial targets since the in vivo milieu is fundamentally different. Mutant derivatives of a clinical isolate of Acinetobacter baumannii were screened for growth on human ascites, an ex vivo medium that reflects the infection environment. A subset of 34 mutants with unique gene disruptions that demonstrated little to no growth on ascites underwent evaluation in a rat subcutaneous abscess model, establishing 18 (53%) of these genes as in vivo essential. The putative gene products all had annotated biological functions, represented unrecognized or underexploited antimicrobial targets, and could be grouped into five functional categories: metabolic, two-component signaling systems, DNA/RNA synthesis and regulation, protein transport, and structural. These A. baumannii in vivo essential genes overlapped poorly with the sets of essential genes from other Gram-negative bacteria catalogued in the Database of Essential Genes (DEG), including those of Acinetobacter baylyi, a closely related species. However, this finding was not due to the absence of orthologs. None of the 18 in vivo essential genes identified in this study, or their putative gene products, were targets of FDA-approved drugs or drugs in the developmental pipeline, indicating that a significant portion of the available target space within pathogenic Gram-negative bacteria is currently neglected. IMPORTANCE: The human pathogen Acinetobacter baumannii is of increasing clinical importance, and a growing proportion of isolates are multiantimicrobial-resistant, pan-antimicrobial-resistant, or extremely resistant strains. This scenario is reflective of the general problem of a critical lack of antimicrobials effective against antimicrobial-resistant Gram-negative bacteria, such as Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter sp., and Escherichia coli. This study identified a set of A. baumannii genes that are essential for growth and survival during infection and demonstrated the importance of using clinically relevant media and in vivo validation while screening for essential genes for the purpose of developing new antimicrobials. Furthermore, it established that if a gene is absent from the Database of Essential Genes, it should not be excluded as a potential antimicrobial target. Lastly, a new set of high-value potential antimicrobial targets for pathogenic Gram-negative bacteria has been identified.

Biofilm formed by a hypervirulent (hypermucoviscous) variant of Klebsiella pneumoniae does not enhance serum resistance or survival in an in vivo abscess model.

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. It has been shown that hvKP strains produce more biofilm than "classical" stains of K. pneumoniae, therefore we hypothesized that biofilm formation may contribute to the pathogenesis of systemic infection. To test this hypothesis, transposon mutants of the model pathogen hvKP1 were generated and screened for decreased production of biofilm. Three mutant constructs with disruptions in glnA [putatively encodes glutamine synthetase, hvKP1 glnA:: EZ::TN < KAN-2 > (glnA::Tn)], sucD [putatively encodes succinyl-CoA synthase α subunit, hvKP1 sucD:: EZ::TN < KAN-2 > (sucD::Tn)], and tag [putatively encodes transcriptional antiterminator of glycerol uptake operon, hvKP1 tag:: EZ::TN < KAN-2 > (tag::Tn)] were chosen for further characterization and use in biologic studies. Quantitative assays performed in rich laboratory medium and human ascites confirmed the phenotype and a hypermucoviscosity assay established that capsule production was not affected. However, compared with its wild-type parent, neither planktonic cells nor biofilms of glnA::Tn, sucD::Tn and tag::Tn displayed a change to the bactericidal activity of 90% human serum. Likewise, when assessed in a rat subcutaneous abscess model, the growth and survival of glnA::Tn, sucD::Tn and tag::Tn in abscess fluid was similar to hvKP1. In this report we identify three new genes that contribute to biofilm formation in hvKP1. However, decreased biofilm production due to disruption of these genes does not affect the sensitivity of these mutant constructs to 90% human serum when in planktonic form or within a biofilm. Further, their virulence in an in vivo abscess model was unaffected.

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.

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 ß-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.

The K1 capsular polysaccharide of Acinetobacter baumannii strain 307-0294 is a major virulence factor.

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.

Identification and characterization of a glycosyltransferase involved in Acinetobacter baumannii lipopolysaccharide core biosynthesis.

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.

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.