Specific bacterial commensals demonstrating multidrug resistance (MDR) are opportunistic pathogens for immunocompromised patients, including Corynebacterium species (spp.). Severe infections due to MDR corynebacteria are being increasingly reported where several MDR phenotypes have been described. One such phenotype, the macrolide-lincosamide-streptogramin B phenotype (MLSB), is characterized by high-level resistance to macrolides, lincosamides, and streptogramin B. Resistance is thought to be attributable to acquisition of the ermX gene, a methyltransferase that alters the ribosomal macrolide binding site. Until recently, ermX had been reported in only six Corynebacterium spp. We have observed other corynebacteria can also display high-level resistance to MLSB antimicrobials and are ermX positive. Hypotheses being tested include: 1) high-level macrolide and lincosamide resistance in Corynebacterium spp. is caused by acquiring ermX; 2) distribution of ermX is more widespread than previously published; 3) ermX is associated with transposon Tn5432; 4) multidrug resistance has spread to Canadian C. afermentans and C. aurimucosum strains.

Thesis (Ph. D.)--Rutgers University, 2007. / "Graduate Program in Computational Biology and Molecular Biophysics." Includes bibliographical references (p. 86-94).

Thesis (Ph. D.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains ix, 219 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 212-219).

Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: Hung-wen Liu. Vita. Includes bibliographical references. Available also from UMI company.

Thesis (Ph. D.)--University of California, San Diego, 1999. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

<p>Antibiotic resistance is often linked to human use of antibiotics. However, antibiotics and antibiotic biosynthetic pathways have been evolving for millions of years suggesting that antibiotic resistance is an ancient phenomenon. As of now, there has been no systematic survey of environmental microbes proven to exist in the absence of human influence and Lechuguilla cave offers such environment<em>. </em> Resistance diversity in strains isolated from this cave was analyzed by a phenotypic screen against a panel of 26 different antibiotics. Resistant strains were further investigated through determination of minimal inhibitory concentration (MIC) and inactivation studies. Of particular interest was strain LC044 (<em>Brachybacterium paraconglomeratum</em>), observed to inactivate macrolide antibiotics by phosphorylation. Genome sequencing and bioinformatics (BLAST analysis) identified a putative macrolide phosphotransferase (MPH) in strain LC044 and biochemical characterization of the purified recombinant protein confirmed its macrolide inactivating properties. To investigate if characterized MPH was unique to cave isolate, available terrestrial <em>Brachybacterium faecium</em> DSM 4810 genome was mined for presence of MPH-like protein. The top hit to the MPH from LC044 (a protein with 282 amino acids and 72% identity) was heterologously expressed and purified. Complete biochemical analysis of this enzyme revealed (i) MPH-activity, despite its annotation as aminoglycoside phosphotransferase (APH), and (ii) no significant differences in substrate specificities or kinetic parameters between these two enzymes suggesting that these two enzymes were equally effective resistance enzymes. This work highlights the prevalence of antibiotic resistance in a pristine, cave ecosystem and provides further support for the theory that antibiotic resistance is everywhere. Furthermore, the <em>mph</em> resistance determinant found in cave isolate and closely related terrestrial isolate show homology to clinical<em> mph</em> genes, suggesting that environmental <em>mph</em> genes could have served as reservoir of clinical determinants.</p> / Master of Science (MSc)

The soil-dwelling gram-positive coccobacillus Rhodococcus equi is a well-known veterinary pathogen and emerging human pathogen. Although Rhodococcus infection is primarily associated with pyogranulomatous pneumonia in foals, these bacteria can also infect other animal species including humans. R. equi pathogenicity is mediated by the conjugative virulence plasmid (pVAP) which promotes intracellular proliferation in host macrophages. Currently R. equi is endemic in horse breeding farms worldwide. No R. equi vaccine is available and both treatment and prophylaxis rely on the administration of a prolonged course with a combination of macrolide antibiotics (typically erythromycin) and rifampicin. These antimicrobials were introduced in the therapy against R. equi in the 1980s and multiresistance has now emerged among foal isolates, increasing the risk of zoonotic transmission. In this thesis, the role of conjugal extrachromosomal replicons in the host adaptation of R. equi was explored. A previous epidemiological study indicated that different variants of the pVAP virulence plasmid are associated with different animal hosts. This PhD project provides experimental confirmation of a R. equi plasmid-driven host tropism. In vivo and in vitro competition assays were performed using a set of isogenic strains only different in the virulence plasmid type, in adapted (horse) and non-adapted (mouse) model species. The data obtained in the horse model provides clear evidence of a significant negative selection of the non-equine virulence plasmids both at a cell and at the animal level, while no selection was observed in the non-adapted mouse model. Furthermore, this project characterized the determinant responsible for macrolide resistance in R. equi, a novel erm methylase gene, erm(46). The erm(46) determinant was shown to be transferable between strains by conjugation and herein the underlying mechanism and how erm(46) becomes stabilized in R. equi is described. PacBio SMRT-sequencing based analysis revealed that the erm(46) gene is carried in a self-replicating conjugative plasmid of about 80 kb, that we designated pRErm46. The conjugation machinery of pRErm46 was hypothesized to be responsible for bringing the erm(46) determinant into R. equi. However, some erythromycin resistant isolates lack pRErm46 but erm(46) transfer is still observed. This reflects the observation that erm(46) is present in a mobile element that, upon acquisition with the pRErm46 replicon, transposes at a high frequency and to multiple locations of the host genome. If the erm(46) mobile element transposes to the chromosome, no further transfer of the resistance is observed at a detectable frequency in the absence of pRErm46. On the other hand, if the erm(46) element transposes to the R. equi virulence plasmid, the erm(46) determinant co-opts the pVAPA conjugal transfer machinery and gets transferred at the same high frequency as the virulence plasmid (10-2). This constitutes a unique example of efficient co-transfer, in the same genetic vehicle, of virulence and antimicrobial determinants, two key niche-adaptive traits required for within-host survival of bacterial pathogen.

Abstract not available

Chronic rhinosinusitis is a common disorder of chronic inflammation of the upper respiratory tract. It is associated with significant symptoms and impairment of the quality of life of sufferers. Despite recent advances in the medical and surgical management of chronic rhinosinusitis, there remains a population of patients who fail to obtain relief from their symptoms. Chronic inflammation of the mucosa of the nasal cavity and paranasal sinuses is one of the hallmarks of chronic rhinosinusitis. This inflammation is demonstrated by an increased number of chronic inflammatory cells, elevated levels of pro-inflammatory cytokines, increased expression of adhesion molecules and metaplastic changes in the epithelium. The current medical treatments for chronic sinusitis aim to reduce this inflammation and consequently improve symptoms. In recent years, evidence has emerged that macrolide antibiotics have an anti-inflammatory effect that is separate from their anti-bacterial effect. This effect was first described in the treatment of diffuse panbronchiolitis, a disorder of chronic inflammation of the lower respiratory tract. Following the success of macrolides in treating this condition it was trialed in chronic rhinosinusitis. Several open-label trials have subsequently demonstrated a beneficial effect. Laboratory studies have investigated the mechanism of the anti-inflammatory effect of macrolides. These have shown that macrolides effect cytokine production, inflammatory cell apoptosis, expression of adhesion molecules, neutrophil oxidative burst, bacterial virulence and mucociliary function. In this thesis we report a series of experiments designed to further investigate the mechanism of action and clinical effect of macrolides. In vitro studies using whole sections of chronic rhinosinusitis mucosa cultured for 24 hours in macrolide, prednisolone or control showed that macrolide and prednisolone produced significant reductions in the production of interleukin-5, interleukin-8 and granulocyte-macrophage colony stimulating factor. The same cultured specimens also showed a reduction in expression of transforming growth factor-?. No reduction was seen in the expression of the key pro-inflammatory nuclear transcription factor Nuclear factor-?B. In our in vivo experiments, biopsies were taken from chronic rhinosinusitis patients who had received a 3-month course of macrolide. These biopsies showed a reduction in the number of neutrophils present following treatment. There was no reduction in the number of other inflammatory cells or in the expression of TGF-? and NK-?B. We have performed the first ever double-blinded, randomized, placebo-controlled trial of macrolide in the treatment of chronic rhinosinusitis. Patients receiving macrolide showed significant improvements in saccharine transit time, nasal endoscopic scoring and symptom scores following a 12 week course. Patients with low levels of serum immunoglobulin E showed significantly improved outcomes compared to those with high levels. Interleukin-8 levels in nasal lavage fluid were significantly reduced in the patients with low levels of IgE following macrolide treatment. No improvements in any of the objective or subjective outcome measures were seen in the placebo-treated patients. We have performed a series of experiments investigating the anti-inflammatory effect of macrolide antibiotics from 'the bench to the bedside'. These experiments have provided insight into the mechanism of action of macrolides in the laboratory setting and evidence of a beneficial effect in the treatment of chronic rhinosinusitis patients.