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.

Siderophore-mediated iron acquisition by Klebsiella pneumoniae.

Microbes synthesize and secrete siderophores, that bind and solubilize precipitated or otherwise unavailable iron in their microenvironments. Gram (-) bacterial TonB-dependent outer membrane receptors capture the resulting ferric siderophores to begin the uptake process. From their similarity to fepA, the structural gene for the Escherichia coli ferric enterobactin (FeEnt) receptor, we identified four homologous genes in the human and animal ESKAPE pathogen Klebsiella pneumoniae (strain Kp52.145). One locus encodes IroN (locus 0027 on plasmid pII), and three other loci encode other FepA orthologs/paralogs (chromosomal loci 1658, 2380, and 4984). Based on the crystal structure of E. coli FepA (1FEP), we modeled the tertiary structures of the K. pneumoniae FepA homologs and genetically engineered individual Cys substitutions in their predicted surface loops. We subjected bacteria expressing the Cys mutant proteins to modification with extrinsic fluorescein maleimide (FM) and used the resulting fluorescently labeled cells to spectroscopically monitor the binding and transport of catecholate ferric siderophores by the four different receptors. The FM-modified FepA homologs were nanosensors that defined the ferric catecholate uptake pathways in pathogenic strains of K. pneumoniae. In Kp52.145, loci 1658 and 4984 encoded receptors that primarily recognized and transported FeEnt; locus 0027 produced a receptor that principally bound and transported FeEnt and glucosylated FeEnt (FeGEnt); locus 2380 encoded a protein that bound ferric catecholate compounds but did not detectably transport them. The sensors also characterized the uptake of iron complexes, including FeGEnt, by the hypervirulent, hypermucoviscous K. pneumoniae strain hvKp1. IMPORTANCE: Both commensal and pathogenic bacteria produce small organic chelators, called siderophores, that avidly bind iron and increase its bioavailability. Klebsiella pneumoniae variably produces four siderophores that antagonize host iron sequestration: enterobactin, glucosylated enterobactin (also termed salmochelin), aerobactin, and yersiniabactin, which promote colonization of different host tissues. Abundant evidence links bacterial iron acquisition to virulence and infectious diseases. The data we report explain the recognition and transport of ferric catecholates and other siderophores, which are crucial to iron acquisition by K. pneumoniae.

Anatomy of an extensively drug-resistant Klebsiella pneumoniae outbreak in Tuscany, Italy.

A protracted outbreak of New Delhi metallo-ß-lactamase (NDM)-producing carbapenem-resistant Klebsiella pneumoniae started in Tuscany, Italy, in November 2018 and continued in 2020 and through 2021. To understand the regional emergence and transmission dynamics over time, we collected and sequenced the genomes of 117 extensively drug-resistant, NDM-producing K. pneumoniae isolates cultured over a 20-mo period from 76 patients at several healthcare facilities in southeast Tuscany. All isolates belonged to high-risk clone ST-147 and were typically nonsusceptible to all first-line antibiotics. Albeit sporadic, resistances to colistin, tigecycline, and fosfomycin were also observed as a result of repeated, independent mutations. Genomic analysis revealed that ST-147 isolates circulating in Tuscany were monophyletic and highly genetically related (including a network of 42 patients from the same hospital and sharing nearly identical isolates), and shared a recent ancestor with clinical isolates from the Middle East. While the blaNDM-1 gene was carried by an IncFIB-type plasmid, our investigations revealed that the ST-147 lineage from Italy also acquired a hybrid IncFIB/IncHIB-type plasmid carrying the 16S methyltransferase armA gene as well as key virulence biomarkers often found in hypervirulent isolates. This plasmid shared extensive homologies with mosaic plasmids circulating globally including from ST-11 and ST-307 convergent lineages. Phenotypically, the carriage of this hybrid plasmid resulted in increased siderophore production but did not confer virulence to the level of an archetypical, hypervirulent K. pneumoniae in a subcutaneous model of infection with immunocompetent CD1 mice. Our findings highlight the importance of performing genomic surveillance to identify emerging threats.

Fluorescent sensors of siderophores produced by bacterial pathogens.

Siderophores are iron-chelating molecules that solubilize Fe3+ for microbial utilization and facilitate colonization or infection of eukaryotes by liberating host iron for bacterial uptake. By fluorescently labeling membrane receptors and binding proteins, we created 20 sensors that detect, discriminate, and quantify apo- and ferric siderophores. The sensor proteins originated from TonB-dependent ligand-gated porins (LGPs) of Escherichia coli (Fiu, FepA, Cir, FhuA, IutA, BtuB), Klebsiella pneumoniae (IroN, FepA, FyuA), Acinetobacter baumannii (PiuA, FepA, PirA, BauA), Pseudomonas aeruginosa (FepA, FpvA), and Caulobacter crescentus (HutA) from a periplasmic E. coli binding protein (FepB) and from a human serum binding protein (siderocalin). They detected ferric catecholates (enterobactin, degraded enterobactin, glucosylated enterobactin, dihydroxybenzoate, dihydroxybenzoyl serine, cefidericol, MB-1), ferric hydroxamates (ferrichromes, aerobactin), mixed iron complexes (yersiniabactin, acinetobactin, pyoverdine), and porphyrins (hemin, vitamin B12). The sensors defined the specificities and corresponding affinities of the LGPs and binding proteins and monitored ferric siderophore and porphyrin transport by microbial pathogens. We also quantified, for the first time, broad recognition of diverse ferric complexes by some LGPs, as well as monospecificity for a single metal chelate by others. In addition to their primary ferric siderophore ligands, most LGPs bound the corresponding aposiderophore with ∼100-fold lower affinity. These sensors provide insights into ferric siderophore biosynthesis and uptake pathways in free-living, commensal, and pathogenic Gram-negative bacteria.

Penicillin Binding Protein 7/8 Is a Potential Drug Target in Carbapenem-Resistant Acinetobacter baumannii.

Limited therapeutic options dictate the need for new classes of antimicrobials active against carbapenem-resistant Acinetobacter baumannii. Presented data confirm and extend penicillin binding protein 7/8 (PBP 7/8) as a high-value target in the CR A. baumannii strain HUMC1. PBP 7/8 was essential for optimal growth/survival of HUMC1 in ex vivo human ascites and in a rat subcutaneous abscess model; in a mouse pneumonia model, the absence of PBP 7/8 decreased lethality 11-fold. The loss of PBP 7/8 resulted in increased permeability, sensitivity to complement, and lysozyme-mediated bactericidal activity. These changes did not appear to be due to alterations in the cellular fatty acid composition or capsule production. However, a decrease in lipid A and an increase in coccoidal cells and cell aggregation were noted. The compromise of the stringent permeability barrier in the PBP 7/8 mutant was reflected by an increased susceptibility to several antimicrobials. Importantly, expression of ampC was not significantly affected by the loss of PBP 7/8 and serial passage of the mutant strain in human ascites over 7 days did not yield revertants possessing a wild-type phenotype. In summary, these data and other features support PBP 7/8 as a high-value drug target for extensively drug-resistant and CR A. baumannii. Our results guide next-stage studies; the determination that the inactivation of PBP 7/8 results in an increased sensitivity to lysozyme enables the design of a high-throughput screening assay to identify small molecule compounds that can specifically inhibit PBP 7/8 activity.

Capsule carbohydrate structure determines virulence in Acinetobacter baumannii.

Acinetobacter baumannii is a highly antibiotic-resistant bacterial pathogen for which novel therapeutic approaches are needed. Unfortunately, the drivers of virulence in A. baumannii remain uncertain. By comparing genomes among a panel of A. baumannii strains we identified a specific gene variation in the capsule locus that correlated with altered virulence. While less virulent strains possessed the intact gene gtr6, a hypervirulent clinical isolate contained a spontaneous transposon insertion in the same gene, resulting in the loss of a branchpoint in capsular carbohydrate structure. By constructing isogenic gtr6 mutants, we confirmed that gtr6-disrupted strains were protected from phagocytosis in vitro and displayed higher bacterial burden and lethality in vivo. Gtr6+ strains were phagocytized more readily and caused lower bacterial burden and no clinical illness in vivo. We found that the CR3 receptor mediated phagocytosis of gtr6+, but not gtr6-, strains in a complement-dependent manner. Furthermore, hypovirulent gtr6+ strains demonstrated increased virulence in vivo when CR3 function was abrogated. In summary, loss-of-function in a single capsule assembly gene dramatically altered virulence by inhibiting complement deposition and recognition by phagocytes across multiple A. baumannii strains. Thus, capsular structure can determine virulence among A. baumannii strains by altering bacterial interactions with host complement-mediated opsonophagocytosis.

Hypervirulent Klebsiella pneumoniae.

Hypervirulent K. pneumoniae (hvKp) is an evolving pathotype that is more virulent than classical K. pneumoniae (cKp). hvKp usually infects individuals from the community, who are often healthy. Infections are more common in the Asian Pacific Rim but are occurring globally. hvKp infection frequently presents at multiple sites or subsequently metastatically spreads, often requiring source control. hvKp has an increased ability to cause central nervous system infection and endophthalmitis, which require rapid recognition and site-specific treatment. The genetic factors that confer hvKp's hypervirulent phenotype are present on a large virulence plasmid and perhaps integrative conjugal elements. Increased capsule production and aerobactin production are established hvKp-specific virulence factors. Similar to cKp, hvKp strains are becoming increasingly resistant to antimicrobials via acquisition of mobile elements carrying resistance determinants, and new hvKp strains emerge when extensively drug-resistant cKp strains acquire hvKp-specific virulence determinants, resulting in nosocomial infection. Presently, clinical laboratories are unable to differentiate cKp from hvKp, but recently, several biomarkers and quantitative siderophore production have been shown to accurately predict hvKp strains, which could lead to the development of a diagnostic test for use by clinical laboratories for optimal patient care and for use in epidemiologic surveillance and research studies.

Fact versus Fiction: a Review of the Evidence behind Alcohol and Antibiotic Interactions.

Many antibiotics carry caution stickers that warn against alcohol consumption. Data regarding concurrent use are sparse. An awareness of data that address this common clinical scenario is important so health care professionals can make informed clinical decisions and address questions in an evidence-based manner. The purpose of this systematic review was to determine the evidence behind alcohol warnings issued for many common antimicrobials. The search was conducted from inception of each database to 2018 using PubMed, Medline via Ovid, and Embase. It included studies that involved interactions, effects on efficacy, and toxicity/adverse drug reactions (ADR) due to concomitant alcohol consumption and antimicrobials. All interactions were considered in terms of three components: (i) alteration in pharmacokinetics/pharmacodynamics (PK/PD) of antimicrobials and/or alcohol, (ii) change in antimicrobial efficacy, and (iii) development of toxicity/ADR. Available data support that oral penicillins, cefdinir, cefpodoxime, fluoroquinolones, azithromycin, tetracycline, nitrofurantoin, secnidazole, tinidazole, and fluconazole can be safely used with concomitant alcohol consumption. Data are equivocal for trimethoprim-sulfamethoxazole. Erythromycin may have reduced efficacy with alcohol consumption, and doxycycline may have reduced efficacy in chronic alcoholism. Alcohol low in tyramine may be consumed with oxazolidinones. The disulfiram-like reaction, though classically associated with metronidazole, occurs with uncertain frequency and with varied severity. Cephalosporins with a methylthiotetrazole (MTT) side chain or a methylthiodioxotriazine (MTDT) ring, ketoconazole, and griseofulvin have an increased risk of a disulfiram-like reaction. Alcohol and antimicrobial interactions are often lacking evidence. This review questions common beliefs due to poor, often conflicting data and identifies important knowledge gaps.

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.

Getting hypervirulent Klebsiella pneumoniae on the radar screen.

PURPOSE OF REVIEW: Two pathotypes of Klebsiella pneumoniae cause human infections, classical (cKp) and hypervirulent (hvKp) K. pneumoniae. The present understanding of genetic elements, the need for an accurate test to identify hvKp, the clinical implications of infection, the knowledge gap on how and why hvKp colonization transitions to infection, and potential infection prevention and control issues for hvKp are discussed. RECENT FINDINGS: Infections because of hvKp are increasingly recognized worldwide. Its ability to cause organ and life-threatening disease in healthy individuals from the community merits concern, which has been magnified by increasing descriptions of multiply drug-resistant (MDR) and extensively drug-resistant (XDR) strains. Increased capsule and siderophore production by hvKp relative to cKp are critical virulence traits. Asians are most commonly infected, but whether this is mediated by a genetic susceptibility, or increased exposure and colonization is unknown. Specific studies about the epidemiology and transmission of hvKp are lacking, but precautions are appropriate for MDR/XDR strains and perhaps all infected/colonized individuals. SUMMARY: hvKp is evolving into an increasingly concerning pathogen, in part because of the development of XDR strains. An accurate test to identify hvKp is needed for optimal clinical care, epidemiological, and research studies. An improved understanding of how infection develops, if a genetic susceptibility exists, and appropriate infection prevention and control measures also are needed.

Monoclonal Antibody Protects Against Acinetobacter baumannii Infection by Enhancing Bacterial Clearance and Evading Sepsis.

Background: Extremely drug-resistant (XDR) Acinetobacter baumannii is one of the most commonly encountered, highly resistant pathogens requiring novel therapeutic interventions. Methods: We developed C8, a monoclonal antibody (mAb), by immunizing mice with sublethal inocula of a hypervirulent XDR clinical isolate. Results: C8 targets capsular carbohydrate on the bacterial surface, enhancing opsonophagocytosis. Treating with a single dose of C8 as low as 0.5 µg/mouse (0.0167 mg/kg) markedly improved survival in lethal bacteremic sepsis and aspiration pneumonia models of XDR A. baumannii infection. C8 was also synergistic with colistin, substantially improving survival compared to monotherapy. Treatment with C8 significantly reduced blood bacterial density, cytokine production (tumor necrosis factor α, interleukin [IL] 6, IL-1ß, and IL-10), and sepsis biomarkers. Serial in vitro passaging of A. baumannii in the presence of C8 did not cause loss of mAb binding to the bacteria, but did result in emergence of less-virulent mutants that were more susceptible to macrophage uptake. Finally, we developed a highly humanized variant of C8 that retains opsonophagocytic activity in murine and human macrophages and rescued mice from lethal infection. Conclusions: We describe a promising and novel mAb as therapy for lethal, XDR A. baumannii infections, and demonstrate that it synergistically improves outcomes in combination with antibiotics.

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 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.

Polymyxin B in Combination with Rifampin and Meropenem against Polymyxin B-Resistant KPC-Producing Klebsiella pneumoniae.

Safe and effective therapies are urgently needed to treat polymyxin-resistant KPC-producing Klebsiella pneumoniae infections and suppress the emergence of resistance. We investigated the pharmacodynamics of polymyxin B, rifampin, and meropenem alone and as polymyxin B-based double and triple combinations against KPC-producing K. pneumoniae isolates. The rates and extents of killing with polymyxin B (1 to 128 mg/liter), rifampin (2 to 16 mg/liter), and meropenem (10 to 120 mg/liter) were evaluated against polymyxin B-susceptible (PBs) and polymyxin B-resistant (PBr) clinical isolates using 48-h static time-kill studies. Additionally, humanized triple-drug regimens of polymyxin B (concentration at steady state [Css] values of 0.5, 1, and 2 mg/liter), 600 mg rifampin every 12 or 8 h, and 1 or 2 g meropenem every 8 h dosed as an extended 3-h infusion were simulated over 48 h by using a one-compartment in vitro dynamic infection model. Serial bacterial counts were performed to quantify the pharmacodynamic effect. Population analysis profiles (PAPs) were used to assess the emergence of polymyxin B resistance. Monotherapy was ineffective against both isolates. Polymyxin B with rifampin demonstrated early bactericidal activity against the PBs isolate, followed by regrowth by 48 h. Bactericidal activity was sustained at all polymyxin B concentrations of ≥2 mg/liter in combination with meropenem. No two-drug combinations were effective against the PBr isolate, but all simulated triple-drug regimens showed early bactericidal activity against both strains by 8 h that was sustained over 48 h. PAPs did not reveal the emergence of resistant subpopulations. The triple-drug combination of polymyxin B, rifampin, and meropenem may be a viable consideration for the treatment of PBr KPC-producing K. pneumoniae infections. Further investigation is warranted to optimize triple-combination therapy.

Important Complexities of the Antivirulence Target Paradigm: A Novel Ostensibly Resistance-Avoiding Approach for Treating Infections.

Use of antivirulence therapy has assumed that inhibition of bacterial fitness at the site of infection without directly affecting viability will minimize the development of resistance. However, selection for resistant strains is much more likely to occur at sites of colonization or in the environment following excretion of the therapeutic agent. Data are needed regarding whether the drug's target promotes fitness among bacteria in (drug-exposed) niches other than sites of infection. Furthermore, in vivo studies of resistance selection should assess off-target selection for resistance (eg, within the microbiome). Only when such data are available can the risk for development of resistance be gauged appropriately.

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.

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.