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.

The purpose of this research was to characterize the Escherichia coli, E. coli, allantoinase enzyme. Allantoinase catalyses the conversion allantoin to allantoate via the hydrolysis of a cyclic amide bond and is coded for by the allB gene. The enzyme is a member of the amidohydrolase superfamily. Amidohydrolase superfamily enzymes have a common (&#945;&#946;)8-barrel structure but catalyze the hydrolysis of many different substrates by a common mechanism. The structural characteristics and roles of divalent cations of enzymes in this superfamily will be discussed and related to previous work conducted on allantoinases. In this work, the metal dependence of allantoinase was initially studied by Mn, Co, Zn, Cd, and Ni-supplemented assays of enzyme of very low metal content. By changing the growth conditions under which the allB was overexpressed in E. coli, and the addition of Zn, Co or Mn to the culture, enzyme with bound Zn (ZnALN), Co (CoALN) or Mn (MnALN) was produced. The pH dependence of log (kcat/KM) for allantoinase in the presence of MnCl2, ZnALN and CoALN followed a bell-shaped curve, indicating that one ionizable group needed to be deprotonated and the deprotonation of a second group caused a decrease in catalytic activity. The pK1 for ionization at low pH was dependent upon which divalent cation was present and is concluded to be that of the deprotonation of water. A structural model of allantoinase with bound allantoin was constructed and used to determine which amino acid residues may be involved in catalysis. Allantoinase mutants R67K, C152A, C152S, C287A, C287S, S317A, D315N and W332F were purified. The kinetic parameters kcat, KM and kcat/KM of wild type and mutant allantoinases were compared. The possible roles of these amino acid residues in catalysis and substrate binding, and the results of the pH rate profiles are discussed. A catalytic mechanism for allantoinase is proposed.

Con el objetivo de determinar los serotipos más frecuente de Escherichia coli Enteropatógena (EPEC) en los cuadros diarreicos agudos de niños menores de cinco años, se diseñó una investigación de tipo descriptivo, transversal, y observacional. La población de estudio estuvo constituida por 251 niños tratados en forma ambulatoria en el Hospital Nacional Docente Madre Niño “San Bartolomé” durante los meses de noviembre del 2000 a marzo del 2001. Se encontró 42 % (105 casos) de coprocultivos positivos a bacterias enteropatógenas, siendo Escherichia coli enteropatógena (EPEC) con 29,2 % (35 casos), la segunda causa más frecuente después de Shigella sp. Los serotipos de EPEC encontrados fueron: 9 casos del O119, 8 casos de O26, 6 casos del O55 y 2 casos del O86. Se concluye que lo serotipos más frecuentes de EPEC en los cuadros diarreicos agudos de niños menores de cinco años fueron el O119 y el O26.

Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Includes bibliographical references (leaves 149-164). Available also in a digital version from Dissertation Abstracts.

Thèse doctorat : Ecologie Microbienne : Villeurbanne, INSA : 2005. / Titre provenant de l'écran-titre. Bibliogr. p. 107-121.