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

Les systèmes toxine-antitoxine (TA) bactériens ont été découverts il y a une vingtaine d’année sur les plasmides à bas nombre de copie. Ils sont composés de deux gènes organisés en opéron, l’un codant pour une toxine stable et l’autre pour une antitoxine instable capable de neutraliser l’effet de la toxine. Les systèmes TA sont fortement représentés au sein de l’ensemble des génomes bactériens. Ils se localisent aussi bien sur des éléments génétiques mobiles (plasmides, phages, transposons,…) que dans les chromosomes, ce qui suggère que le transfert horizontal de gènes participe à leur dissémination. Le système TA ccd du plasmide F d’Escherichia coli (ccdF) est composé de l’antitoxine CcdA et de la toxine CcdB. Le système ccdF contribue à la stabilité du plasmide F en tuant les bactéries-filles n’ayant pas reçu de copies plasmidiques lors de la division bactérienne (tuerie post-ségrégationelle). Au cours de ce travail, nous avons caractérisé un homologue du système toxine-antitoxine ccd du plasmide F (ccdF) qui se situe dans le chromosome de la souche pathogène E. coli O157:H7 EDL933 entre les gènes folA et apaH (ccdO157). Les systèmes ccdF et ccdO157 coexistent naturellement dans les souches d’E. coli O157:H7, le système ccdF se trouvant sur le plasmide pO157 qui dérive du plasmide F. Nos résultats montrent que l’antitoxine plasmidique CcdAF neutralise l’effet de la toxine chromosomique CcdBO157, tandis que l’antitoxine chromosomique CcdAO157 ne contrecarre pas la toxicité de la toxine plasmidique CcdBF. Nous avons également montré que le système ccdF cause une tuerie post-ségrégationelle, lorsqu’il est cloné dans un plasmide instable, dans une souche possédant le système chromosomique ccdO157. Le système ccdF est donc fonctionnel en présence de son homologue chromosomique. Le système ccdO157 est absent du chromosome de la souche de laboratoire E. coli K-12 MG1655, où une région intergénique de 77 pb sépare les gènes folA et apaH. Celle-ci contient une séquence cible pour la transposition. Nous avons étudié la distribution du système ccdO157 au sein de 523 souches d’E. coli représentatives de l’ensemble des sérogroupes décrits. Nos résultats montrent que le système ccdO157 est présent au sein de souches appartenant à 47 sérogroupes différents. Nos résultats mettent en évidence la diversité de la région intergénique folA-apaH d’E. coli. Celle-ci peut contenir gènes codant pour des protéines présentant de l’homologie avec des protéines d’espèce bactériennes éloignées d’E. coli ou d’organismes eucaryotes, ainsi qu’un élément génétique mobile, l’IS621, ce qui montre que le système ccdO157 a intégré le chromosome d’E. coli via le transfert horizontal de gènes.

Stafylococcus aureus, Stafylococcus epidermidis, Escherichia coli, Streptococcus pyogenes och Bacillus sp. är bakterier som förekommer i vår normalflora eller runt om i vår omgivning. De är i de allra flesta fall harmlösa men kan orsaka sjukdom ifall de hamnar i t.ex. sår eller kontaminerar mat. Vissa av dessa bakterier har även en tendens att utveckla resistens mot antibiotika, infektioner orsakade av resistenta bakterier kan bli allvarliga och väldigt svåra att behandla. Detta är det främsta skälet till varför vi har valt att undersöka ifall de ovan nämnda bakterierna förkommer på avdelningen SET på Halmstad högskola. För att kunna identifiera de bakterier vi fann använde vi oss av en rad olika biokemiska tester och resistensbestämningen utfördes bl.a. med hjälp av en PCR körning. Vi fann alla de bakterier vi sökte efter förutom S. pyogenes och ingen av de funna visade på någon resistens mot antibiotika.

Ribosomes are multicomponent macromolecular particles and are essential for the survival of cells in all organisms. The ribosome's universal function is to catalyze polypeptide synthesis through translation of mRNA transcripts. Ribosomes from Escherichia coli, eubacterial organisms, have a sedimentation coefficient of 70S and are composed of 30S and 50S ribonucleoprotein subunits. The small ribosomal subunit is an assembly of 21 different proteins and a 16S ribosomal RNA. Within the 16S rRNA there are a few short stretches of universally conserved sequences spanning positions 517-533, 1394-1408, and 1492-1506. Clear functions for these sequence zones have not yet been assigned. Here I report a kinetic analysis of these highly conserved regions in the 16S rRNA and within the 30S ribosomal subunits. Binding affinity was measured in experiments that were based on protection from nuclease 51 digestion of short oligodeoxynucleotides hybridized to the designated regions. DNAs hybridized to regions 1400 and 1500 show significant differences in the apparent dissociation constants when measured in 30S particles as opposed to those found for 16S rRNA. Region 525 showed no difference in kinetic behavior. To further elucidate the functional and structural role played by the region centered about C1400 in 16S rRNA, a four nucleotide deletion was constructed within this region. The deletion was introduced by direct RNA manipulation using DNA/RNA hybridization, RNase H digestions, and ligation of the correct RNA fragments with T4 RNA ligase. I improved ligation efficiency of large RNA molecules by including a connector looped short DNA oligomer. Recycling products through phenyl boronate agarose (PBA-30) column also improved the efficiency of ligation. The mutagenized 16S rRNA fully reassembles into 30 particles and the altered 30S subunit possesses all of the normal ribosomal proteins. Altered ribosomes were functional in in vitro translation of MS2 mRNA. The altered ribosomes have lower translational activity relative to controls. Here I present indirect evidence suggesting that the decrease in the synthesis of MS2 coat proteins is the result of premature termination. The altered 16S RNA in ribosomes had an apparent dissociation constants with DNA probes comparable to those found for normal 16S rRNA. This suggest that the RNA is less flexible in the particle relative to normal 30S subunits. The deletion at 1400 did not have any effect on the physical properties of the 1500 region, as measured by DNA hybridization. A minor, but significant, effect on the 525 region was observed. A possible RNA/RNA interaction within the 30S particle is proposed to account for this observation. / Graduation date: 1992

The purpose of this study was to investigate the prevalence and mechanisms of extended-spectrum cephalosporin- (ESC) and carbapenem-resistance in Escherichia coli from the GTA. A total of 526 non-duplicate E. coli clinical isolates were collected during March 1-5, 2010 from 13 participant hospitals. Among these, 71 isolates showed reduced susceptibility (rS, intermediate, and/or resistant phenotype) to cefoxitin (FOX) and/or ESC. No carbapenem resistance was detected. Extended-spectrum ß-lactamase genes detected (n=37; 52.1%) belong to the CTX-M-family, including blaCTX-M-15 (78.4%), blaCTX-M-23 (2.7%), blaCTX-M-14 (18.9%). The only plasmid-mediated ampC gene identified among FOXrS isolates (n=49; 69%) was blaCMY-2 (n=7; 14.3%). Seventeen strains (24%) were negative for all ß-lactamase genes tested. Analysis of the chromosomal ampC promoter revealed mutations associated with AmpC hyperproduction. Other mechanisms of resistance (e.g. impermeability and/or unidentified ß-lactamases) cannot be discarded. The most prevalent clone detected was ST131. IncFIA, FIB and Frep were the most common plasmid replicon types detected.

The purpose of this study was to investigate the prevalence and mechanisms of extended-spectrum cephalosporin- (ESC) and carbapenem-resistance in Escherichia coli from the GTA. A total of 526 non-duplicate E. coli clinical isolates were collected during March 1-5, 2010 from 13 participant hospitals. Among these, 71 isolates showed reduced susceptibility (rS, intermediate, and/or resistant phenotype) to cefoxitin (FOX) and/or ESC. No carbapenem resistance was detected. Extended-spectrum ß-lactamase genes detected (n=37; 52.1%) belong to the CTX-M-family, including blaCTX-M-15 (78.4%), blaCTX-M-23 (2.7%), blaCTX-M-14 (18.9%). The only plasmid-mediated ampC gene identified among FOXrS isolates (n=49; 69%) was blaCMY-2 (n=7; 14.3%). Seventeen strains (24%) were negative for all ß-lactamase genes tested. Analysis of the chromosomal ampC promoter revealed mutations associated with AmpC hyperproduction. Other mechanisms of resistance (e.g. impermeability and/or unidentified ß-lactamases) cannot be discarded. The most prevalent clone detected was ST131. IncFIA, FIB and Frep were the most common plasmid replicon types detected.

E. coli thioredoxin (TRX) was modified by the episulfonium ion derived from S-(2-chloroethyl)glutathione (CEG) or S-(2-chloroethyl)cysteine (CEC). The alkylation site was located at Cys-32, which was confirmed by tandem mass spectrometry. Two forms of native TRX, Oxi- and Red-TRX, and two modified TRXs, GS- and Cys-TRX, were examined by hydrogen/deuterium (H/D) exchange reactions using electrospray ionization mass spectrometry (ESI-MS) for the analysis. Conformational dynamics during thermal denaturation were probed by H/D exchange-in experiments. Under conditions in which the folded conformational state is marginally stable, H/D exchange-in experiments resulted in mass spectra differing in the number of incorporated deuteriums which indicates the presence of two distinct populations of molecules. As the exchange-in time increased, the population representing the unfolded state increased and the population for the folded state decreased. The rate of conversion was used to estimate the rate constant of unfolding. ESI mass spectra were also recorded as a function of temperature without H/D exchange, and the observed bimodal charge state distributions were analyzed in order to estimate melting temperatures. GS-TRX showed increased resistance to hydrogen isotope exchange in comparison with Red-TRX indicating that there were enhanced intramolecular interactions in the former protein. Pepsin digestion was performed on deuterated TRXs to analyze different structural regions. The amount of deuterium incorporated was monitored with peptic peptides from deuterated TRXs with different exchange-in incubation periods. Deuterium levels of each peptide were plotted versus the exchange time and fitted with a series of first-order rate terms. The regions 59-80 and 81-108 of Oxi- and Red-TRX showed an EX1 mechanism as evidenced by two distinct mass envelopes that appeared after a short incubation time in deuterated solvent. Tandem mass spectrometry (MS/MS) was carried out to obtain the information on individual amide linkages. MS/MS data showed generally excellent correlations with the exchange rate constants from published NMR data on Oxi- and Red-TRXs. Two residues, Ile-75 and Ala-93 in GS-TRX indicated the most probable sites responsible for induced H-bonding by the attached glutathionyl group, which was consistent with the energy minimized structure predicted by AMBER force field constants. / Graduation date: 2001

This study evaluated methods for controlling Escherichia coli O157:H7 and Salmonella non-pathogenic bacterial surrogates during the production of marinated non-intact beef products. Hot (~30 degrees C) boneless, beef strip loins (n = 54, Institutional Meat Purchase Specification 180) were inoculated with one of two levels (approximately 5.8 and 1.9 log10 CFU/cm2, hereafter referred to as high- and low-inoculated, respectively) of non-pathogenic, rifampicin-resistant E. coli organisms used to simulate harvest floor contamination. The inoculated beef strip loins were chilled at 2 degrees C for 24 h, and then vacuum packaged and aged for 7 to 24 days at 2 degrees C. The beef strip loins were subjected to one of five treatments or control (no treatment). Spray treatments were: 2.5% L-lactic acid, 5.0% L-lactic acid, 1,050 ppm acidified sodium chlorite, 205 ppm peroxyacetic acid, and tap water. Lactic acid treatments were applied at ~53 degrees C, whereas the other sprays were applied at room temperature (~25 degrees C). Treated and control pieces were tumble marinated using a commercial marinade. Sample counts were collected throughout the experiment to track reductions in inoculated microorganisms as impacted by antimicrobial treatment and processing. For the high-inoculated strip loins, the 5.0% L-lactic acid treatment was most effective (P < 0.05) across treatments and control at reducing surrogate organisms on meat surfaces before marination, producing a 2.6 log10 CFU/cm2 reduction. The water treatment accounted for the least (P < 0.05) reductions across treatments and control of surrogate organisms on the meat surface before marination. Peroxyacetic acid produced the greatest reduction of surface surrogate organisms in the finished, marinated product. The water treatment resulted in greater internalization of surrogate microorganisms when compared to the control. Furthermore, certain less effective antimicrobial sprays such as water may facilitate internalization of surface bacteria, more so than non-treated subprimals. It is important that producers of non-intact beef products focus on using effective antimicrobial sprays that maximize reductions and minimize internalization of surface bacteria into the finished product.

Over the last few decades, increased urbanization has led to a new recognition in stream health – the urban stream or the urban stream syndrome. Understanding urban water quality is important for identifying those factors or sources that contribute to impairment. Many streams are listed as impaired because of the increased concentrations of pathogens. While wastewater treatment plants (WWTPs) discharge effluent that has been disinfected, often downstream from WWTPs point sources are high numbers of indicator bacteria, Escherichia coli. This study collected data on the recovery and regrowth of E. coli by collecting ultraviolet light treated effluent from the Carters Creek WWTP and spiked it with three different concentrations of DOC derived from a leaf and grass extract. Escherichia coli were enumerated at 6, 12, 18, 24, 36, 48, and 72 hours. After 6 h growth for each of the grass treatments, except for the control and high grass treatment exceeded the primary contact recreation standard for surface water quality. At 18h the low and high leaf treatments exceeded the primary contact recreation standard for surface water quality. The chemistry of each flask was analyzed for DOC, total N, NO3-N, NH4-N, Na , K , Mg 2, Ca 2, F-, Cl-, SO4-2 and PO4-3 at t=0 and t=72 h. CNP values for both leaf and grass treatments ranged from 2.22 - 36.5. Regrowth was not observed in those treatments with a CNP value below 5. Biodegradability of the treatments was examined to identify the limiting nutrient. By focusing on reducing the CNP value below 5 of the receiving water, recovery and regrowth of E. coli downstream from WWTPs can be reduced. The biodegradability test suggested that in the presence of excess DOC, N is the limiting nutrient. Certain macroinvertebrate species, Ephermeroptera, Trichoptera, and Plecoptera (EPT), are indicators of good stream health. Macroinvertebrates were collected at nine watersheds within the Bryan/College Station area, a rapidly urbanizing community, upstream and downstream from WWTPs and analyzed for relative abundance of pollution intolerant (percent EPT) and pollution tolerant species. All sites downstream from a WWTP had percent EPT present in the collection.

Environmental sources within 5 feedlots were sampled for E. coli O157:H7 and O157:NM to determine the prevalence of this pathogen with a view to minimize or control its spread in the feedlot environment. Monthly samples were taken from the feedlots in the Panhandle and South Plains of Texas over a nine-month period. Samples were examined by an immunomagnetic bead separation, followed by plating onto CT-SMAC and CHROMagar O157 media. Sorbitol-negative colonies were tested using ImmunoCard Stat! E. coli O157:H7 Plus and confirmed as E. coli O157:H7, using biochemical (Vitek system) and serological tests (latex agglutination). Additionally, one hundred sponge samples were collected from the hides of stunned cattle at the slaughter plant. All isolates were subjected to rep-PCR DNA fingerprinting and antimicrobial profiling. E. coli O157 was isolated from hide (56%) and environmental samples (4%). E. coli O157 was isolated from all environmental sources, with peak prevalence during November (9%) and March (10%). At least one sample from each feedlot was positive 42% of the time. The most contaminated sites were the chute area (6%) and sludge from waste water ponds (6%). Positive samples were most frequently found from feedlot 5 (7%) and the greatest variation in positive samples between feedlots (0-34%) occurred during March. A decrease in the presence of E. coli O157 in feedlots was observed during January (0%), when ambient, water, and pond sludge temperatures were consistently low. No correlation with other environmental factors was observed. Hide was a primary source of E. coli O157 on carcasses with an overall prevalence of 56%. Of two sampling days, the number of positive hide samples varied from 14% for the first day to 98% for the second day. The total positive samples collected (environmental (47); hide (56)) were 64% H7, and 36% NM. The environmental isolates showed similar antibiotic resistance patterns, regardless of the source. Most E. coli O157 isolates from the feedlots and hides showed a high level of resistance to cephalothin (45%) and sulfisoxazole (56%). E. coli O157 isolates from feedlots were resistant to more than 10 antibiotics (9/317). All of the isolates appeared highly similar, with an average similarity of 53% by rep-PCR DNA fingerprinting.