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.

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Summary Hfq is an RNA‐binding protein that functions in post‐transcriptional gene regulation by mediating interactions between mRNAs and small regulatory RNAs (sRNAs). Two proteins encoded by BAB1_1794 and BAB2_0612 are highly over‐produced in a Brucella abortus hfq mutant compared with the parental strain, and recently, expression of orthologues of these proteins in Agrobacterium tumefaciens was shown to be regulated by two sRNAs, called AbcR1 and AbcR2. Orthologous sRNAs (likewise designated AbcR1 and AbcR2) have been identified in B. abortus 2308. In Brucella, abcR1 and abcR2 single mutants are not defective in their ability to survive in cultured murine macrophages, but an abcR1 abcR2 double mutant exhibits significant attenuation in macrophages. Additionally, the abcR1 abcR2 double mutant displays significant attenuation in a mouse model of chronic Brucella infection. Quantitative proteomics and microarray analyses revealed that the AbcR sRNAs predominantly regulate genes predicted to be involved in amino acid and polyamine transport and metabolism, and Northern blot analyses indicate that the AbcR sRNAs accelerate the degradation of the target mRNAs. In an Escherichia coli two‐plasmid reporter system, overexpression of either AbcR1 or AbcR2 was sufficient for regulation of target mRNAs, indicating that the AbcR sRNAs from B. abortus 2308 perform redundant regulatory functions.

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Methicillin-resistant Staphylococcus aureus is estimated to cause more U.S. deaths annually than HIV/AIDS. The emergence of hypervirulent and multidrug-resistant strains has further amplified public health concern and accentuated the need for new classes of antibiotics. RNA degradation is a required cellular process that could be exploited for novel antimicrobial drug development. However, such discovery efforts have been hindered because components of the Gram-positive RNA turnover machinery are incompletely defined. In the current study we found that the essential S. aureus protein, RnpA, catalyzes rRNA and mRNA digestion in vitro. Exploiting this activity, high through-put and secondary screening assays identified a small molecule inhibitor of RnpA-mediated in vitro RNA degradation. This agent was shown to limit cellular mRNA degradation and exhibited antimicrobial activity against predominant methicillin-resistant S. aureus (MRSA) lineages circulating throughout the U.S., vancomycin intermediate susceptible S. aureus (VISA), vancomycin resistant S. aureus (VRSA) and other Gram-positive bacterial pathogens with high RnpA amino acid conservation. We also found that this RnpA-inhibitor ameliorates disease in a systemic mouse infection model and has antimicrobial activity against biofilm-associated S. aureus. Taken together, these findings indicate that RnpA, either alone, as a component of the RNase P holoenzyme, and/or as a member of a more elaborate complex, may play a role in S. aureus RNA degradation and provide proof of principle for RNA catabolism-based antimicrobial therapy.

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.

BackgroundComputational analysis of metagenomes requires the taxonomical assignment of the genome contigs assembled from DNA reads of environmental samples. Because of the diverse nature of microbiomes, the length of the assemblies obtained can vary between a few hundred bp to a few hundred Kbp. Current taxonomic classification algorithms provide accurate classification for long contigs or for short fragments from organisms that have close relatives with annotated genomes. These are significant limitations for metagenome analysis because of the complexity of microbiomes and the paucity of existing annotated genomes.ResultsWe propose a robust taxonomic classification method, RAIphy, that uses a novel sequence similarity metric with iterative refinement of taxonomic models and functions effectively without these limitations. We have tested RAIphy with synthetic metagenomics data ranging between 100 bp to 50 Kbp. Within a sequence read range of 100 bp-1000 bp, the sensitivity of RAIphy ranges between 38%-81% outperforming the currently popular composition-based methods for reads in this range. Comparison with computationally more intensive sequence similarity methods shows that RAIphy performs competitively while being significantly faster. The sensitivity-specificity characteristics for relatively longer contigs were compared with the PhyloPythia and TACOA algorithms. RAIphy performs better than these algorithms at varying clade-levels. For an acid mine drainage (AMD) metagenome, RAIphy was able to taxonomically bin the sequence read set more accurately than the currently available methods, Phymm and MEGAN, and more accurately in two out of three tests than the much more computationally intensive method, PhymmBL.ConclusionsWith the introduction of the relative abundance index metric and an iterative classification method, we propose a taxonomic classification algorithm that performs competitively for a large range of DNA contig lengths assembled from metagenome data. Because of its speed, simplicity, and accuracy RAIphy can be successfully used in the binning process for a broad range of metagenomic data obtained from environmental samples.