Codon usage comparison of novel genes in clinical isolates of Haemophilus influenzae

A similarity statistic for codon usage was developed and used to compare novel gene sequences found in clinical isolates of Haemophilus influenzae with a reference set of 80 prokaryotic, eukaryotic and viral genomes. These analyses were performed to obtain an indication as to whether individual genes were Haemophilus-like in nature, or if they probably had more recently entered the H.influenzae gene pool via horizontal gene transfer from other species. The average and SD values were calculated for the similarity statistics from a study of the set of all genes in the H.influenzae Rd reference genome that encoded proteins of 100 amino acids or longer. Approximately 80% of Rd genes gave a statistic indicating that they were most like other Rd genes. Genes displaying codon usage statistics >1 SD above this range were either considered part of the highly expressed group of H.influenzae genes, or were considered of foreign origin. An alternative determinant for identifying genes of foreign origin was when the similarity statistics produced a value that was much closer to a non-H.influenzae reference organism than to any of the Haemophilus species contained in the reference set. Approximately 65% of the novel sequences identified in the H.influenzae clinical isolates displayed codon usages most similar to Haemophilus sp. The remaining novel sequences produced similarity statistics closer to one of the other reference genomes thereby suggesting that these sequences may have entered the H.influenzae gene pool more recently via horizontal transfer

Death and transfiguration in static staphylococcus epidermidis cultures

The overwhelming majority of bacteria live in slime embedded microbial communities termed biofilms, which are typically adherent to a surface. However, when several Staphylococcus epidermidis strains were cultivated in static liquid cultures, macroscopic aggregates were seen floating within the broth and also sedimented at the test tube bottom. Light- and electron microscopy revealed that early-stage aggregates consisted of bacteria and extracellular matrix, organized in sheetlike structures. Perpendicular under the sheets hung a network of periodically arranged, bacteria-associated strands. During the extended cultivation, the strands of a subpopulation of aggregates developed into cross-connected wall-like structures, in which aligned bacteria formed the walls. The resulting architecture had a compartmentalized appearance. In late-stage cultures, the wall-associated bacteria disintegrated so that, henceforth, the walls were made of the coalescing remnants of lysed bacteria, while the compartment-like organization remained intact. At the same time, the majority of strand containing aggregates with associated culturable bacteria continued to exist. These observations indicate that some strains of Staphylococcus epidermidis are able to build highly sophisticated structures, in which a subpopulation undergoes cell lysis, presumably to provide continued access to nutrients in a nutrient-limited environment, whilst maintaining structural integrity

Virulence phenotypes of low-passage clinical isolates of Nontypeable Haemophilus influenzae assessed using the chinchilla laniger model of otitis media

<p>Abstract</p> <p>Background</p> <p>The nontypeable Haemophilus influenzae (NTHi) are associated with a spectrum of respiratory mucosal infections including: acute otitis media (AOM); chronic otitis media with effusion (COME); otorrhea; locally invasive diseases such as mastoiditis; as well as a range of systemic disease states, suggesting a wide range of virulence phenotypes. Genomic studies have demonstrated that each clinical strain contains a unique genic distribution from a population-based supragenome, the distributed genome hypothesis. These diverse clinical and genotypic findings suggest that each NTHi strain possesses a unique set of virulence factors that contributes to the course of the disease.</p> <p>Results</p> <p>The local and systemic virulence patterns of ten genomically characterized low-passage clinical NTHi strains (PittAA – PittJJ) obtained from children with COME or otorrhea were stratified using the chinchilla model of otitis media (OM). Each isolate was used to bilaterally inoculate six animals and thereafter clinical assessments were carried out daily for 8 days by blinded observers. There was no statistical difference in the time it took for any of the 10 NTHi strains to induce otologic (local) disease with respect to any or all of the other strains, however the differences in time to maximal local disease and the severity of local disease were both significant between the strains. Parameters of systemic disease indicated that the strains were not all equivalent: time to development of the systemic disease, maximal systemic scores and mortality were all statistically different among the strains. PittGG induced 100% mortality while PittBB, PittCC, and PittEE produced no mortality. Overall Pitt GG, PittII, and Pitt FF produced the most rapid and most severe local and systemic disease. A post hoc determination of the clinical origins of the 10 NTHi strains revealed that these three strains were of otorrheic origin, whereas the other 7 were from patients with COME.</p> <p>Conclusion</p> <p>Collectively these data suggest that the chinchilla OM model is useful for discriminating between otorrheic and COME NTHi strains as to their disease-producing potential in humans, and combined with whole genome analyses, point the way towards identifying classes of virulence genes.</p

Bacterial plurality as a general mechanism driving persistence in chronic infections

Classical methods for the study of bacterial pathogens have proven to be inadequate to inform with respect to chronic infections including those associated with arthroplasties. Modern methods of analysis have demonstrated that bacterial growth patterns, ecology, and intra-species heterogeneity are more complex than were envisioned by early microbiologists. Cultural methods were developed to study acute, epidemic infections, but it is now recognized that the phenotype associated with these diseases represents only a minor aspect of the bacterial life cycle, which consists of planktonic, attachment, biofilm, and dispersal phases. Over 99% of bacteria in natural populations are found in biofilms which contain multiple ecological niches and numerous phenotypes. Unfortunately, the effort to develop antibiotics has been directed solely at the planktonic minority (associated with systemic illness) which explains our inability to eradicate chronic infections. In this study we establish a new rubric, bacterial plurality, for the understanding of bacterial ecology and evolution with respect to chronic infection. The fundamental tenets of bacterial plurality are that the bacteria within an infecting population display multiple phenotypes and possess multiple genotypes. Phenotypic plurality is embodied in the biofilm paradigm and genotypic plurality is embodied in the concepts of the supra-genome and the distributed genome hypothesis. It is now clear that bacterial diversity provides bacterial populations, as a whole, the ability to persist in the face of a multi-faceted host response

Cellulosilyticum ruminicola, a Newly Described Rumen Bacterium That Possesses Redundant Fibrolytic-Protein-Encoding Genes and Degrades Lignocellulose with Multiple Carbohydrate- Borne Fibrolytic Enzymes▿ †

Cellulosilyticum ruminicola H1 is a newly described bacterium isolated from yak (Bos grunniens) rumen and is characterized by its ability to grow on a variety of hemicelluloses and degrade cellulosic materials. In this study, we performed the whole-genome sequencing of C. ruminicola H1 and observed a comprehensive set of genes encoding the enzymes essential for hydrolyzing plant cell wall. The corresponding enzymatic activities were also determined in strain H1; these included endoglucanases, cellobiohydrolases, xylanases, mannanase, pectinases, and feruloyl esterases and acetyl esterases to break the interbridge cross-link, as well as the enzymes that degrade the glycosidic bonds. This bacterium appears to produce polymer hydrolases that act on both soluble and crystal celluloses. Approximately half of the cellulytic activities, including cellobiohydrolase (50%), feruloyl esterase (45%), and one third of xylanase (31%) and endoglucanase (36%) activities were bound to cellulosic fibers. However, only a minority of mannase (6.78%) and pectinase (1.76%) activities were fiber associated. Strain H1 seems to degrade the plant-derived polysaccharides by producing individual fibrolytic enzymes, whereas the majority of polysaccharide hydrolases contain carbohydrate-binding module. Cellulosome or cellulosomelike protein complex was never isolated from this bacterium. Thus, the fibrolytic enzyme production of strain H1 may represent a different strategy in cellulase organization used by most of other ruminal microbes, but it applies the fungal mode of cellulose production

Autophagy Protects Against Senescence and Apoptosis via the RAS-Mitochondria in High-Glucose-Induced Endothelial Cells

Backgrounds: Autophagy is an important process in the pathogenesis of diabetes and plays a critical role in maintaining cellular homeostasis. However, the autophagic response and its mechanism in diabetic vascular endothelium remain unclear. Methods and Results: We studied high-glucose-induced renin-angiotensin system (RAS)-mitochondrial damage and its effect on endothelial cells. With regard to therapeutics, we investigated the beneficial effect of angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin II type 1 receptor blockers (ARBs) against high-glucose-induced endothelial responses. High glucose activated RAS, enhanced mitochondrial damage and increased senescence, apoptosis and autophagic-responses in endothelial cells, and these effects were mimicked by using angiotensin II (Ang). The use of an ACEI or ARB, however, inhibited the negative effects of high glucose. Direct mitochondrial injury caused by carbonyl cyanide 3-chlorophenylhydrazone (CCCP) resulted in similar negative effects of high glucose or Ang and abrogated the protective effects of an ACEI or ARB. Additionally, by impairing autophagy, high-glucose-induced senescence and apoptosis were accelerated and the ACEI- or ARB-mediated beneficial effects were abolished. Furthermore, increases in FragEL™ DNA Fragmentation (TUNEL)-positive cells, β-galactosidase activation and the expression of autophagic biomarkers were revealed in diabetic patients and rats, and the treatment with an ACEI or ARB decreased these responses. Conclusions: These data suggest that autophagy protects against senescence and apoptosis via RAS-mitochondria in high-glucose-induced endothelial cells

Engineering approaches for the detection and control of orthopaedic biofilm infections

Artificial joints are subject to chronic infections associated with bacterial biofilms, which only can be eradicated by the traumatic removal of the implant followed by sustained intravenous antibiotic therapy. We have adopted an engineering approach to develop electrical-current-based approaches to bacterial eradication and microelectromechanical systems that could be embedded within the implanted joint to detect the presence of bacteria and to provide in situ treatment of the infection before a biofilm can form. In the former case we will examine the combined bactericidal effects of direct and indirect electrical fields in combination with antibiotic therapy. In the latter case, bacterial detection will occur by developing a microelectromechanical-systems-based biosensor that can "eavesdrop" on bacterial quorum-sensing-based communication systems. Treatment will be effected by the release of a cocktail of pharmaceutical reagents contained within integral reservoirs associated with the implant, including a molecular jamming signal that competitively binds to the bacteria's quorum sensing receptors (which will "blind" the bacteria, preventing the production of toxins) and multiple high dose antibiotics to eradicate the planktonic bacteria. This approach is designed to take advantage of the relatively high susceptibility to antibiotics that planktonic bacteria display compared with biofilm envirovars. Here we report the development of a generic microelectromechanical systems biosensor that measures changes in internal viscosity in a base fluid triggered by a change in the external environment