Growth of Staphylococcus aureus with the NSAID salicylate increases phenotypic resistance (SAPAR), and the frequency at which heritable resistance occurs to various antibiotics (SAGAR). This study describes the effect of salicylate on heritable and phenotypic resistance to a set of antibiotics for laboratory and multi-drug resistant strains of S. aureus and investigates the link between resistance and SAGAR. Drug gradient plates were used to determine phenotypic resistance to antibiotics targeting DNA replication, transcription, translation and the cell wall in the presence or absence of salicylate. To measure heritable resistance, mutation frequencies were determined for each antibiotic in the presence and absence of salicylate. Salicylate significantly increased mutation frequency of SH1000 to ciprofloxacin 27- fold from 4.9 x 10-8 to 8.5 x 10-7. A significant 8.5- fold increase was observed for LAC from 5.2 x 10-7 to 2.1 x 10-6. Conversely, salicylate significantly decreased mutation frequency for SH1000 to lincomycin 0.035-fold from 3.4 x 10-7 to 1.3 x 10-7. Deletion of the general stress sigma factor sigB encoding σB in SH1000 resulted in decreased heritable and phenotypic resistance, signifying the importance of σB in the full expression of both phenotypes. Metabolite profiling revealed downregulation of glycolysis, TCA, pentose phosphate pathway, and amino acid metabolism. The downregulation of the TCA cycle was confirmed as observed through an increase in NAD+ at growth toxic concentrations of salicylate. Salicylate has been shown to result in ROS accumulation and disruption of proton motive force in mitochondria. SAGAR was only detected for fluoroquinolones, which have been shown to impair TCA cycle and result in ROS accumulation. Examination of ROS under growth-toxic concentrations of salicylate did not reveal a significant increase in ROS levels. Also, the combination of ciprofloxacin and salicylate did not result in an increase in ROS levels. Despite this, addition of the antioxidant glutathione abrogated SAGAR for ciprofloxacin in SH1000 but not for SAPAR. Analysis of SAGAR with NSAIDs benzoate and acetyl salicylic acid revealed a necessity for the ortho hydroxyl group on salicylate to fully express SAGAR. These results suggest that salicylate has pleiotropic effects on S. aureus that include antimicrobial resistance, altered metabolic flux and accumulation of ROS as well as unidentified regulatory genes.

β-barrel membrane proteins play important structural and functional roles in Gram negative bacteria and in mitochondria and chloroplasts of eukaryotes. A conserved machine is responsible for the folding and insertion of β-barrel membrane proteins but its mechanism remains largely unknown. In E. coli, a five protein β-barrel assembly machine (Bam) assembles β-barrel proteins into the outer membrane (OM). Among all β-barrel membrane proteins in E. coli , the β-barrel component of the OM LPS translocon is one of only two essential β-barrels, the other being the central component of the Bam machinery itself. The OM LPS translocon, which consists of OM β-barrel protein LptD (lipopolysaccharide transport) and OM lipoprotein LptE, is responsible for the final export of LPS molecules into the outer leaflet of the OM, resulting in an asymmetric bilayer that blocks the entry of toxic molecules such as antibiotics. This thesis describes the characterization of the biogenesis pathway of the OM LPS translocon and its folding and insertion into the OM by the Bam machinery. An in vivo S35-Methionine pulse-labeling assay was developed to identify intermediates along the biogenesis of the OM LPS translocon. Seven intermediates were identified along the pathway. We show that proper assembly of the OM LPS translocon involves an oxidative disulfide bond rearrangement from a nonfunctional intermediate containing non-native disulfides. We also found that the rate determining step of OM LPS translocon biogenesis is β-barrels folding process by the Bam machinery. Using in vivo chemical crosslinking, we accumulated and trapped a mutant form of LptD on BamA, the central component of the Bam machinery. We extended the S35-Methionine pulse-labeling method to allow chemical crosslinking of substrates on the Bam complex and trapped LptD while it was being folded on the Bam machine. We demonstrated that the interaction between LptD and BamA is independent of LptE, while that between LptD and BamD, the other essential component of the Bam complex beside BamA, is LptE dependent. Based on these findings, we proposed a model of Bam-assisted folding of the OM LPS translocon in which LptE templates the folding of LptD. / Chemistry and Chemical Biology

<p>Risk factors such as intrauterine and vaginal infection put pregnant women at risk for delivering preterm. Bacterial vaginosis (BV) is a polymicrobial clinical syndrome commonly diagnosed in women of reproductive age, with women of African descent with low socioeconomic status and previous preterm delivery at high risk. Although frequently isolated from healthy women,&nbsp / Gardnerella vaginalis has been most frequently associated with BV. There is limited data available on the prevalence of BV in Southern Africa / therefore, we embarked on a study to determine the&nbsp / prevalence of BV and G. vaginalis in predominantly black communities in the Western Cape, in order to establish the role of G. vaginalis in BV. Women attending various Maternity and Obstetrics&nbsp / units (MOU) in the Cape Peninsula with and without a history of pre-term delivery (PTD) were invited to participate in the study. Several factors were statistically associated with pregnancy history,&nbsp / including location of study population, parity, smoking and presence of clinical symptoms. The presence of G. vaginalis was determined by culture in 51.7% of the preterm delivery group (PTDG)&nbsp / and 44% of the full-term delivery group (FTDG) women. BV was detected in 31.13% of PTDG and 23.67% of FTDG by Gram stained analysis according to Nugent scoring criteria, with age and HIV&nbsp / status posing as risk factors. When comparing PTDG and FTDG for an association between the presence of G. vaginalis and BV, a stronger association was observed in the PTDG but it was not statistically significant. In both PTDG and FTDG, G. vaginalis was isolated significantly more often in women diagnosed with BV at 24.5% (p &lt / 0.05). Antibiogram studies revealed both Metronidazole and Clindamycin resistant strains of G. vaginalis. G. vaginalis Biotype 7 is specifically associated with BV, while Biotype 2 appears to be associated with BV in women with a history&nbsp / of PTD. Accuracy of diagnostic tools were tested and it was determined that Nugent scoring is more sensitive in diagnosing BV (76.04%), but culture for G. vaginalis is more specific (83.21%). Although this study was limited in that we were unable to follow-up pregnancy outcomes, we were able to confirm the perceived role of G. vaginalis in BV.&nbsp / </p>

The ß-lactam antibiotics are the most important antibacterial agents in the treatment of infectious diseases. A severe problem in ß-lactam therapy is the emergence of ß-lactam resistant bacteria. Clinical ß-lactam resistance is most often due to the production of ß-lactamases. ß-lactamase genes reside either on plasmids or on the chromosome. The aim of this study was to acquire an understanding of organisation and regulation of chromosomal ß- lactamase genes in different Gram negative species and to elucidate the mechanisms for ampC hyperproduction in the in vivo situation. By DNA hybridization with an ampC probe from Escherichia coli K-12 it was shown that other Gram negative bacteria contained an artpC like chromosomal gene, suggesting a common evolutionary origin. Furthermore, the preceding frd operon that overlaps the ampC gene in E. coli K-12 was found to be much more conserved than the ampC gene in the bacterial species investigated. The ampC and frd opérons in Shigella sonnei and Citrobacter freundii were cloned and characterized by physical mapping. The respective maps were compared to the ampC and frd region in E. coli K-12. The physical map of Sh. sonnei was almost identical to the E. coli K-12 map, whereas in C. freundii only the frd region exhibited any considerable homology. Moreover, in C. freundii, the anpC and frd regions were separated by 1100 basepairs. It is suggested that this DNA is involved in the induction of ß-lactamase production in this organism. A hypothesis for the evolution of the anpC operon in enterobacteria is presented. By isolating and characterizing six ß-lactam resistant clinica], isolates of E. coli hyperproducing the dhrcmosomal ß-lactamase, genetic mechanisms for in vivo evolved resistance was aimed at. These isolates exhibited a 24-48 fold increase in ß-lactamase production. The ß-lactamase produced was found to be biochemically and immunologically identical to the ß-lactamase produced by E. coli K-12. The ampC control region of these six E. coli isolates was DNA-seqenced. The cause of ß-lactamase hyperproduction in five of the clinical E. coli isolates, identical in the DNA segment sequenced, was due to a strong novel ampC promoter displaced 5 bp upstream of the ampC promoter defined in E. coli K-12. The ß-lactamase hyperproduction in the sixth clinical isolate was shown to be caused by two mutations affecting both the promoter and the attenuator in the regulatory region defined by E. coli K- 12. The obtained changes were sufficient to explain the increase in ampC ß- 1 act ama se expression exhibited in these clinical E. coli isolates. Sequence analysis of the ampC control region in Sh. sonnei revealed that it was, with one exception, identical to the one found in the five clinical E. coli ß-lactamase hyperproducers. The only difference was in a position that creates the strong novel ampC promoter in the E. coli hyperproducers. By isolating spontaneous Sh. sonnei mutants with a 40-fold increase in ß-lactamase production carrying the same novel ampC promoter as the clinical E. coli isolates it was concluded that this DNA segment has been transferred in vivo frcm Shigella to E. coli across the species barrier. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1983; Härtill 5 uppsatser</p> / digitalisering@umu

In practice, most approaches for text error detection and correction are based on a conventional domain-dependent background dictionary that represents a fixed and static collection of correct words of a given language and, as a result, satisfactory correction can only be achieved if the dictionary covers most tokens of the underlying correct text. Again, most approaches for text correction are for only one or at best a very few types of errors. The purpose of this thesis is to propose an unsupervised approach to detecting and correcting text errors, that can compete with supervised approaches and answer the following questions: Can an unsupervised approach efficiently detect and correct a text containing multiple errors of both syntactic and semantic nature? What is the magnitude of error coverage, in terms of the number of errors that can be corrected? We conclude that (1) it is possible that an unsupervised approach can efficiently detect and correct a text containing multiple errors of both syntactic and semantic nature. Error types include: real-word spelling errors, typographical errors, lexical choice errors, unwanted words, missing words, prepositional errors, article errors, punctuation errors, and many of the grammatical errors (e.g., errors in agreement and verb formation). (2) The magnitude of error coverage, in terms of the number of errors that can be corrected, is almost double of the number of correct words of the text. Although this is not the upper limit, this is what is practically feasible. We use engineering approaches to answer the first question and theoretical approaches to answer and support the second question. We show that finding inherent properties of a correct text using a corpus in the form of an n-gram data set is more appropriate and practical than using other approaches to detecting and correcting errors. Instead of using rule-based approaches and dictionaries, we argue that a corpus can effectively be used to infer the properties of these types of errors, and to detect and correct these errors. We test the robustness of the proposed approach separately for some individual error types, and then for all types of errors. The approach is language-independent, it can be applied to other languages, as long as n-grams are available. The results of this thesis thus suggest that unsupervised approaches, which are often dismissed in favor of supervised ones in the context of many Natural Language Processing (NLP) related tasks, may present an interesting array of NLP-related problem solving strengths.

Ovine (strains 9L and 3384Y) and bovine (strains 649, 2336 and 8025) isolates of Histophilus somni were investigated for their ability to acquire iron from haemoglobin (Hb). Bovine isolates were capable of utilizing bovine, but not ovine, porcine or human Hb as a source of iron. Ovine isolates could not obtain iron from Hb. Bovine isolates bound bovine, ovine, and human Hbs by means of the same iron-repressible receptor(s) and produced a ~120-kDa iron-repressible, outer membrane protein. Using PCR approaches, an iron-regulated operon containing hugX and hugZ homologues and a gene (hgbA) that encodes a TonB-dependent, Hb-binding proteins were identified in strains 649, 9L and 3384Y. In strains 9L and 3384Y, HgbA is truncated offering a possible explanation for their lack of utilization of Hb as an iron source. In strains 2336 and 8025, expression of HgbA was also subject to a form of phase variation.

Las quinolonas son un grupo de antimicrobianos sintéticos, con un amplio espectro de acción y utilizados con gran éxito para el tratamiento de muy diversas patologías. Es también un grupo en plena fase de desarrollo, donde continuamente están apareciendo nuevas moléculas más activas que las preexistentes en muchos casos.Lamentablemente, la resistencia a quinolonas presenta entre la mayoría de sus moléculas lo que se conoce como resistencia cruzada, o susceptibilidad reducida. Por ello y para poder desarrollar nuevas moléculas dentro de esta familia es importante conocer cuales son los mecanismos de resistencia que presentan los microorganismos, en concreto las bacterias Gram-positivas, frente a estos compuestos. De este modo se podrían diseñar nuevas moléculas que no se vean afectadas por los mecanismos de resistencia ya conocidos. Otro de los parámetros importantes es conocer como las propias quinolonas seleccionan la aparición de mutantes resistentes.Por todos estos motivos, para el presente trabajo se plantearon los siguientes objetivos:1- Investigar las bases moleculares de los mecanismos de resistencia en bacterias Gram-positivas. En concreto, a Staphylococcus aureus un importante patógeno nosocomial, a Streptococcus pneumoniae un patógeno extrahospitalario y a Corynebacterium spp. un patógeno oportunista emergente, perteneciente a la flora habitual de la piel.2- Estudiar la selección in vitro de mutantes resistentes a quinolonas en aislamientos clínicos de S. aureus y S. pneumoniae.3- Determinar el grado de mutagenicidad de las diversas fluoroquinolonas y correlacionarla con la selección de mutantes resistentes. / The quinolones constitutes a group of synthetic antimicrobial agents, with broad spectrum, and have been used with great success for the treatment of a wide variety of pathologies. This group of antimicrobials still under development, and continuously, new molecules more active than the pre-existing ones in many cases are appearing. The resistance to quinolones is present between most of their molecules, this phenomenon is called crossed resistance, or reduced susceptibility. For that reason, to develop new molecules within this family, it is important to know the mechanisms of resistance that the microorganisms presented in front of these compounds, in particular in Gram-positive bacteria. In this way, new molecules could be designed to circumvent the mechanisms of resistance already known. Another important parameter is to know how the quinolones select for resistant mutants. By all these reasons, for the present work the following objectives were considered: 1 - To investigate the molecular bases of the mechanisms of resistance in Gram-positive bacteria. In particular, to Staphylococcus aureus an important nosocomial pathogen, Streptococcus pneumoniae a pathogen causing mainly community-acquired infections and to Corynebacterium spp. an emergent opportunistic pathogen, pertaining to the habitual skin flora. 2 - To study the "in vitro" selection of resistant mutants to quinolones in clinical isolates of S. aureus and S. pneumoniae. To determine the power of mutagenicity of different fluoroquinolones and to correlate it with the selection of resistant mutants.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Feb. 19, 2009). Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xv, 139 p.; also includes graphics (some col.) Includes bibliographical references (p. 123-139).

Thesis (M.S.)--Michigan State University. Dept. of Biochemistry and Molecular Biology, 2008. / "Advisor, Todd A. Ciche"--Acknowledgements. Title from PDF t.p. (viewed on Aug. 5, 2009) Includes bibliographical references. Also issued in print.