Acinetobacter baumannii Increases Tolerance to Antibiotics in Response to Monovalent Cations▿ †

Acinetobacter baumannii is well adapted to the hospital environment, where infections caused by this organism are associated with significant morbidity and mortality. Genetic determinants of antimicrobial resistance have been described extensively, yet the mechanisms by which A. baumannii regulates antibiotic resistance have not been defined. We sought to identify signals encountered within the hospital setting or human host that alter the resistance phenotype of A. baumannii. In this regard, we have identified NaCl as being an important signal that induces significant tolerance to aminoglycosides, carbapenems, quinolones, and colistin upon the culturing of A. baumannii cells in physiological NaCl concentrations. Proteomic analyses of A. baumannii culture supernatants revealed the release of outer membrane proteins in high NaCl, including two porins (CarO and a 33- to 36-kDa protein) whose loss or inactivation is associated with antibiotic resistance. To determine if NaCl affected expression at the transcriptional level, the transcriptional response to NaCl was determined by microarray analyses. These analyses highlighted 18 genes encoding putative efflux transporters that are significantly upregulated in response to NaCl. Consistent with this, the effect of NaCl on the tolerance to levofloxacin and amikacin was significantly reduced upon the treatment of A. baumannii with an efflux pump inhibitor. The effect of physiological concentrations of NaCl on colistin resistance was conserved in a panel of multidrug-resistant isolates of A. baumannii, underscoring the clinical significance of these observations. Taken together, these data demonstrate that A. baumannii sets in motion a global regulatory cascade in response to physiological NaCl concentrations, resulting in broad-spectrum tolerance to antibiotics

Broad-spectrum suppression of bacterial pneumonia by aminoglycoside-propagated Acinetobacter baumannii.

Antimicrobial resistance is increasing in pathogenic bacteria. Yet, the effect of antibiotic exposure on resistant bacteria has been underexplored and may affect pathogenesis. Here we describe the discovery that propagation of the human pathogen Acinetobacter baumannii in an aminoglycoside antibiotic results in alterations to the bacterium that interact with lung innate immunity resulting in enhanced bacterial clearance. Co-inoculation of mice with A. baumannii grown in the presence and absence of the aminoglycoside, kanamycin, induces enhanced clearance of a non-kanamycin-propagated strain. This finding can be replicated when kanamycin-propagated A. baumannii is killed prior to co-inoculation of mice, indicating the enhanced bacterial clearance results from interactions with innate host defenses in the lung. Infection with kanamycin-propagated A. baumannii alters the kinetics of phagocyte recruitment to the lung and reduces pro- and anti-inflammatory cytokine and chemokine production in the lung and blood. This culminates in reduced histopathologic evidence of lung injury during infection despite enhanced bacterial clearance. Further, the antibacterial response induced by killed aminoglycoside-propagated A. baumannii enhances the clearance of multiple clinically relevant Gram-negative pathogens from the lungs of infected mice. Together, these findings exemplify cooperation between antibiotics and the host immune system that affords protection against multiple antibiotic-resistant bacterial pathogens. Further, these findings highlight the potential for the development of a broad-spectrum therapeutic that exploits a similar mechanism to that described here and acts as an innate immunity modulator

Inactivation of Phospholipase D Diminishes Acinetobacter baumannii Pathogenesis▿ †

Acinetobacter baumannii is an emerging bacterial pathogen of considerable health care concern. Nonetheless, relatively little is known about the organism's virulence factors or their regulatory networks. Septicemia and ventilator-associated pneumonia are two of the more severe forms of A. baumannii disease. To identify virulence factors that may contribute to these disease processes, genetically diverse A. baumannii clinical isolates were evaluated for the ability to proliferate in human serum. A transposon mutant library was created in a strain background that propagated well in serum and screened for members with decreased serum growth. The results revealed that disruption of A. baumannii phospholipase D (PLD) caused a reduction in the organism's ability to thrive in serum, a deficiency in epithelial cell invasion, and diminished pathogenesis in a murine model of pneumonia. Collectively, these results suggest that PLD is an A. baumannii virulence factor

Identification of an <em>Acinetobacter baumannii</em> Zinc Acquisition System that Facilitates Resistance to Calprotectin-mediated Zinc Sequestration

<div><p><em>Acinetobacter baumannii</em> is an important nosocomial pathogen that accounts for up to 20 percent of infections in intensive care units worldwide. Furthermore, <em>A. baumannii</em> strains have emerged that are resistant to all available antimicrobials. These facts highlight the dire need for new therapeutic strategies to combat this growing public health threat. Given the critical role for transition metals at the pathogen-host interface, interrogating the role for these metals in <em>A. baumannii</em> physiology and pathogenesis could elucidate novel therapeutic strategies. Toward this end, the role for calprotectin- (CP)-mediated chelation of manganese (Mn) and zinc (Zn) in defense against <em>A. baumannii</em> was investigated. These experiments revealed that CP inhibits <em>A. baumannii</em> growth <em>in vitro</em> through chelation of Mn and Zn. Consistent with these <em>in vitro</em> data, Imaging Mass Spectrometry revealed that CP accompanies neutrophil recruitment to the lung and accumulates at foci of infection in a murine model of <em>A. baumannii</em> pneumonia. CP contributes to host survival and control of bacterial replication in the lung and limits dissemination to secondary sites. Using CP as a probe identified an <em>A. baumannii</em> Zn acquisition system that contributes to Zn uptake, enabling this organism to resist CP-mediated metal chelation, which enhances pathogenesis. Moreover, evidence is provided that Zn uptake across the outer membrane is an energy-dependent process in <em>A. baumannii</em>. Finally, it is shown that Zn limitation reverses carbapenem resistance in multidrug resistant <em>A. baumannii</em> underscoring the clinical relevance of these findings. Taken together, these data establish Zn acquisition systems as viable therapeutic targets to combat multidrug resistant <em>A. baumannii</em> infections.</p> </div

CP inhibits <i>A. baumannii</i> growth <i>in vitro</i> through chelation of Mn and Zn.

<p>(<b>A</b>) <i>A. baumannii</i> growth in the presence of increasing concentrations of CP with (dashed lines) or without (solid lines) 25 µM Mn and 25 µM Zn added back. Data represent the average of three biological replicates. (<b>B</b>) Treatment with CP reduces intracellular Mn and Zn accumulation. ICP-MS analyses of intracellular Mn, Fe and Zn expressed as the relative ratio compared to intracellular Cu. ** <i>p</i><0.01, *** <i>p</i><0.001 by two-way ANOVA.</p

Contribution of the Znu system to pathogenesis <i>in vivo</i>.

<p>(<b>A–F</b>) <i>In vivo</i> GFP expression driven by the <i>znu</i> promoter. Frozen sections of lungs harvested at 36 hpi from wildtype (A–C) or S100A9^−/− (D–F) mice infected with WT::<i>p</i>znuGFP and stained with DAPI. Arrowheads in the merged images indicate bacteria expressing GFP. (<b>G–H</b>) Competition infection between wildtype and Δ<i>znuB</i>. Bacterial burden in lungs (<b>G</b>) and livers (<b>H</b>) of mice co-infected with wildtype <i>A. baumannii</i> and Δ<i>znuB</i>. Red symbols indicate CFU below the limit of detection. * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001 as determined by one-way ANOVA.</p

CP contributes to protection against <i>A. baumannii</i> infection.

<p>(<b>A–B</b>) IMS detection of host proteins in lungs harvested at 36 hpi with <i>A. baumannii.</i> Scale bar = 2 cm. (<b>A</b>) Heat map demonstrating the distribution and abundance of S100A8 (<i>m/z</i> = 10,165.8±5 Da) in uninfected wildtype (left) and infected wildtype (middle) or S100A9^−/− (right) mice. (<b>B</b>) Heat map demonstrating the distribution and abundance of a protein exhibiting <i>m/z</i> 5,679±5 Da in uninfected wildtype (left) and infected wildtype (middle) or S100A9^−/− (right) mice. (<b>C–F</b>) Hematoxylin and eosin stained histological sections taken from lungs harvested at 36 hpi from mice infected with <i>A. baumannii</i>. (<b>C, E</b>) Wildtype C57BL/6. (<b>D, F</b>) S100A9^−/−. Scale bars equal 500 µm (C–D) and 50 µm (E–F). Images are representative of sections taken from three mice for each genotype. (<b>G</b>) Survival of wildtype or CP-deficient mice following infection with <i>A. baumannii</i>. Data were combined from three independent experiments with 8–16 mice per group in each experiment. ** <i>p</i><0.01 as determined by Gehan-Breslow-Wilcoxon Test. (<b>H</b>) Bacterial burden in lungs and livers of wildtype or CP-deficient mice 36 hpi with <i>A. baumannii</i>. Data were averaged from three independent experiments with 5–10 mice per group in each experiment. ** <i>p</i><0.01 as determined by Student's <i>t</i> test.</p