Comparative microbial diversity in the gastrointestinal tracts of food animal species

Molecular tools based on small subunit (SSU) rDNA gene sequences offer a powerful and rapid tool for the analysis of complex microbial communities found in the gastrointestinal tracts (GIT) of food animal species. Extensive comparative sequence analysis of SSU rRNA molecules representing a wide diversity of organisms shows that different regions of the molecule vary in sequence conservation. Oligonucleotides complementing regions of universally conversed SSU rRNA sequences are used as universal probes, while those complementing more variable regions of sequence are useful as selective probes targeting species, genus, or phylogenetic groups. Different approaches derive different information and this is highly dependent on the type of target nucleic acid employed and the conceptual and technical basis used for nucleic acid probe design. Generally these approaches can be divided into DNA-based methods employing empirically characterized probes and rRNA-based methods based on comparative sequence analysis for design and interpretation of rational probes. Polymerase chain reaction (PCR) based techniques can also be applied to the analysis of microbial communities in the GIT. Direct cloning of SSU rDNA genes amplified from these complex communities can be used to determine the extent of diversity in these GIT communities. Denaturing gradient gel electrophoresis (DGGE) is another powerful tool for profiling microbial diversity of microbial communities in GI tracts. Sequence analysis of the excised DGGE amplicons can then be used to presumptively identify predominant bacterial species. Examples of how these molecular approaches are being used to study the microbial diversity of communities from steers fed different diets, swine fed probiotics, and Atlantic salmon fed aquaculture diets are presented

In Vitro and In Vivo Activities of PD 0305970 and PD 0326448, New Bacterial Gyrase/Topoisomerase Inhibitors with Potent Antibacterial Activities versus Multidrug-Resistant Gram-Positive and Fastidious Organism Groups

PD 0305970 and PD 0326448 are new bacterial gyrase and topoisomerase inhibitors (quinazoline-2,4-diones) that possess outstanding in vitro and in vivo activities against a wide spectrum of bacterial species including quinolone- and multidrug-resistant gram-positive and fastidious organism groups. The respective MICs (μg/ml) for PD 0305970 capable of inhibiting ≥90% of bacterial strains tested ranged from 0.125 to 0.5 versus staphylococci, 0.03 to 0.06 versus streptococci, 0.25 to 2 versus enterococci, and 0.25 to 0.5 versus Moraxella catarrhalis, Haemophilus influenzae, Listeria monocytogenes, Legionella pneumophila, and Neisseria spp. PD 0326448 MIC(90)s were generally twofold higher versus these same organism groups. Comparative quinolone MIC(90) values were 4- to 512-fold higher than those of PD 0305970. In testing for frequency of resistance, PD 0305970 and levofloxacin showed low levels of development of spontaneous resistant mutants versus both Staphylococcus aureus and Streptococcus pneumoniae. Unlike quinolones, which target primarily gyrA and parC, analysis of resistant mutants in S. pneumoniae indicates that the likely targets of PD 0305970 are gyrB and parE. PD 0305970 demonstrated rapid bactericidal activity by in vitro time-kill testing versus streptococci. This bactericidal activity carried over to in vivo testing, where PD 0305970 and PD 0326448 displayed outstanding Streptococcus pyogenes 50% protective doses (PD(50)s) (oral dosing) of 0.7 and 3.6 mg/kg, respectively (ciprofloxacin and levofloxacin PD(50)s were >100 and 17.7 mg/kg, respectively). PD 0305970 was also potent in a pneumococcal pneumonia mouse infection model (PD(50) = 3.2 mg/kg) and was 22-fold more potent than levofloxacin

A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore

As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from target-based antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidines target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious Gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity