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<p> Multidrug resistant <i>Acinetobacter baumannii,</i> a common etiologic agent of severe nosocomial infections in compromised hosts, usually harbors <i>aac(6&rsquo;)-Ib.</i> This gene codes for an aminoglycoside acetyltransferase that modifies amikacin and other aminoglycosides of clinical relevance. The goal of this work was to interfere with expression of this resistance gene and induce susceptibility to amikacin in resistant pathogens. In vitro translation assays led to the identification of an antisense oligodeoxynucleotide (ODN4) that targets the initiation of translation region of <i>aac(6&rsquo;)-Ib </i> mRNA. An isosequential nuclease-resistant chimeric oligomer composed of 2&rsquo;,4&rsquo;-bridged nucleic acid-NC (BNA^NC) residues and deoxynucleotides (BNA^NC-DNA) covalently bound to the cell-penetrating peptide (RXR)₄XB (where &ldquo;X&rdquo; and &ldquo;B&rdquo; stand for 6-aminohexanoic acid and &beta;-alanine, respectively). This compound, called CPPBD4, inhibited translation at similar levels observed with ODN4. Addition of a combination of Amikacin and CPPBD4 to a culture of an <i>Acinetobacter baumannii</i> clinical strain harboring <i> aac(6&rsquo;)-Ib</i> resulted in growth inhibition indicating that CPPBD4 reached the cytosol and interfere with the expression of the resistance enzyme. </p>

The eukaryotic nucleus is thought to contain an internal protein scaffolding structure which is present in interphase and metaphase cells. We were interested in identifying some of the protein components of the scaffolding structure by isolating proteins which have DNA attached to them (DNA attachment proteins). DNA attachment proteins have been previously described by our group, and are referred to as proteins 1, 2, 3, and 4, respectively. Their apparent molecular weights (Mr) and isoelectric values (pI) are 70,000, 4.3; 70,000, 5.3; 58,000, 5.3; and 57,000, 4.8, respectively. My project was to produce and characterize polyclonal antibodies made against these four proteins. The DNA attachment proteins were injected into rabbits. The serum was affinity purified using Protein-A sepharose chromatography. Antibody preparations 1, 2, and 4 recognized the specific DNA attachment proteins and did not cross react with the others suggesting that the polyclonal sera recognized distinct and separate epitopes.

The efficacy of chloramine in the presence of copper chloride was evaluated for the inactivation of an indicator bacteria Escherichia coli and coliphage MS-2. Both microorganisms were exposed to chloramine with and without copper chloride. Results showed an increase in the inactivation rate of Escherichia coli and MS-2 phage with an increasing concentration of chloramine. To achieve a 99 percent reduction in the number of Escherichia coli, an exposure of 46, 21, 6, and 5 minutes was necessary for 1, 2.5, 5, and 10 mg chloramine/L, respectively. A 99 percent reduction of MS-2 phage occurred after 60 and 25 minutes of exposure to 5 and 10 mg chloramine/L. Chloramine in the presence of copper increased the inactivation rate of Escherichia coli and MS-2 phage. The time needed for 99 percent inactivation of E. coli and MS-2 phage was reduced. Copper increases the inactivation rate of bacteria and viruses by chloramine. (Abstract shortened with permission of author.)

Growth at high temperature of wild isolates of three species of Bacillus was analyzed to assess potential responses to global warming. Experimental populations were grown at temperatures from 32° to 60° C. The higher temperatures include ones near and above maxima previously reported for laboratory strains. Summer soil temperatures, three centimeters below the ground surface, were recorded at the same site from which the wild isolates came, show that temperatures in the Sonoran Desert often reach 50° to 60° C. The growth data show that the desert isolates of B. subtilis and B. licheniformis have thermal maxima close to those reported by Gordon et al. (1973), while B. megaterium grew well at 2-3°C above the reported maximum. Global Climate Models predict a rise of 1° to 4.5°C over the next 60-100 years. Such a rise could shorten periods of active growth and nutrient cycling by Bacillus decomposers.

<p> The struggle for survival between phages and their bacterial hosts is best exemplified by the diverse mechanisms bacteria utilize to block phage infection, and the methods phages use to surmount them. Mycobacteriophage DS6A is unique amongst the more than 8000 identified mycobacteriophages in that its host range is restricted to mycobacteria that are members of the <i><u> My</u>cobacterium <u>t</u>u<u>b</u>erculosis </i> <u>C</u>omplex <i>(MTBC)</i>. However, the molecular mechanism for this specificity remains unknown. To study the relationship DS6A has to both MTBC and non-tuberculous mycobacteria (NTMs), we generated two novel recombinant DS6A shuttle phasmids containing fluorescent reporter mVenus. These phages were utilized to clearly demonstrate that 50 years of previous scientific dogma was incorrect, and DS6A can in fact infect NTM mycobacteria to a wide range of degrees. Work teasing out the mechanisms of resistance is underway. Additionally, we identified the chaperone trigger factor (Tig; Rv2462c) to be required for a productive DS6A infection in <i> Mtb</i>. Interestingly, no host range mutants have been generated that can overcome this resistance to plaguing. Deleting <i>tig</i> did not affect the lysis profile of any of the other >30 mycobacteriophages able to infect <i>Mtb</i>. Susceptibility of <i>Mtb</i> &Delta;<i> tig</i> to DS6A infection was rescued by complementation of tig on an integrating plasmid. DS6A fluorophage infection of <i>Mtb</i> &Delta;<i> tig</i> induced high levels of detectable fluorescence, suggesting Tig is not required for the adsorption of phage or introduction of DS6A DNA. Additionally assays determined that Tig was not involved in transcriptional or translational activation. However, deleting <i>tig</i> did yield an additional phenotype in <i>Mtb</i>; significant attenuation in both immunocompetent and immunocompromised mice. Lipidome and secretome assays showed stark differences between the lipid makeup and secreted protein profiles of the tig mutant versus the WT that could explain the cause of attenuation. For Mtb, the loss of <i> tig</i> is a double edged sword; total resistance to DS6A is afforded in the clonal population, but at the cost of virulence in eukaryotic hosts. </p>