Browsing by Subject "Escherichia coli"
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Item Antibiotic resistance in the lower intestinal microbiota of dairy cattle: longitudinal analysis of phenotypic and genotypic resistance.(2012-02) Boyer, Timothy CharlesThis research focused on methods of measuring antibiotic resistance and analysis of antibiotic resistance data in dairy cattle that were sampled repeatedly over time. Specific objectives included: characterization and longitudinal analysis of phenotypic antibiotic resistance of commensal Escherichia coli, development of a statistical model for the analysis of low quantity resistance genes measured by quantitative real-time polymerase chain reaction (qPCR), measurement of antibiotic resistance genes in the lower intestinal bacterial communities of dairy cattle that received a short-term therapeutic dose of antibiotic and untreated cattle, and measurement and longitudinal analysis of the quantities of six antibiotic resistance genes in the lower intestinal bacterial communities of dairy cattle. Enteric E. coli collected from dairy cattle over 1.5 years were tested for phenotypic resistance to 17 antimicrobials. A total of 93 phenotypic patterns were observed among 3,402 isolates tested, with a majority (67%) susceptible to all 17 antimicrobials. The most prevalent resistances were to tetracycline, sulfamethoxazole, and streptomycin. Latent class and latent transition analyses were carried out to group the animals into classes according to their resistance phenotypes and to estimate the probabilities of transitioning into and out of classes over time. Probabilities of transitioning to a pan-susceptible class were high, as were the probabilities of remaining in the pan-susceptible class. Probabilities of transitioning form a pan-susceptible class to a resistant class were very low. Measurement of antibiotic resistance genes by qPCR presents challenges for genes that are present in very low quantities. A statistical model was developed to analyze qPCR data made up of a significant proportion of observations that fall below the limit of quantification of a qPCR assay. Computer simulations showed that the statistical model produced less biased estimates of regression parameters than common methods of handling low quantity qPCR data. qPCR was applied to a cohort of dairy cattle that received a five day course of ceftiofur and matched untreated cattle. Quantities of a gene (blaCMY-2) that confers resistance to ceftiofur were measured and analyzed using the statistical model developed for low quantity genes. Treated animals had significantly higher quantities of blaCMY-2 during treatment than untreated animals. By the first day post-treatment, gene quantities had returned to pre-treatment levels. The quantities of six different antibiotic resistance genes were measured by qPCR in the fecal community bacterial DNA of a cattle population that was sampled repeatedly over 2.5 years. Significantly increasing trends over time were observed for three of the six genes conferring resistance to tetracyclines, macrolides, and cephalosporins. Comparison of phenotypic resistance and qPCR data showed that qPCR performed on community DNA is a more sensitive method of detection that phenotypic testing of cultured isolates.Item Control of enterohemorrhagic Escherichia coli using bacteriophages(2010-07) Viazis, SteliosEnterohemorrhagic Escherichia coli (EHEC) O157:H7 has been recognized as a major foodborne pathogen responsible for frequent gastroenteritis outbreaks. Phages can be used as a natural antimicrobial method to reduce bacterial pathogens from the food supply. The objective of the first study was to isolate, identify and characterize a diverse collection of lytic bacteriophages capable of infecting EHEC serotypes O26, O111, and O157. Phages were isolated from dairy and feedlot manure using EHEC O157, O26, and O111 strains as hosts. Plaques were purified and screened against additional strains (14, O157; 10, O26; 10, O111) using the efficiency of plating method (EOP). Phage CEV2 and five other phages previously isolated were able to lyse all 14 O157 EHEC strains with EOP values consistently above 0.001. Two phages isolated from fecal slurry from dairy and feedlot cattle were highly effective against strains of E. coli O157, through EOP tests, and against O26 through spot tests, but not O111. Bacterial challenges against high titers of four E. coli O157 strains suggested that a mixture of the 8 most effective phages was just as effective as or more than each individual phage. This collection of phages can be grouped and potentially used as an antimicrobial cocktail to inactivate O157 and O26 serotypes. The objective of the second study was to determine the effect of the bacteriophage cocktail, BEC8, on the viability of a mixture of EHEC O157:H7 strains applied on surfaces of materials representative of food processing plants. Sterile stainless steel chips (SSC), ceramic tile chips (CTC), and high density polyethylene chips (HDPEC) were used. The EHEC O157:H7 strains used were EK27, ATCC 43895, and 472. Exponentially growing cells from tryptic soy (TS) broth cultures were spot inoculated on surfaces and dried. EHEC cells were placed at high, medium, and low inoculum levels (10 6 , 10 5 , and 10 4 CFU/mL, respectively). Appropriate controls and BEC8 (approx. 106 PFU/mL) were applied on treated surfaces. The surfaces were incubated at 4, 12, room temperature (RT), and 37°C. EHEC survival was determined using standard plate count on TS agar. No survivors were detected after BEC8 treatment at a low inoculum level at the following incubation conditions: 37oC for 10 min and RT after 1 h on SSC and CTC; 12°C after 10 min on SSC, 1 h for CTC, and 24 h for HDPEC. These results indicated that the phage cocktail was effective within an hour against low levels of the EHEC mixture at RT on all 3 hard surfaces. The objective of the third study was to determine the effect of the bacteriophage cocktail, BEC8, on its own and in combination with the essential oil trans -cinnameldehyde (TC) on the viability of a mixture of EHEC O157:H7 strains applied on baby romaine lettuce and baby spinach leaves. The EHEC O157:H7 strains used were nalidixic acid resistance mutants of EK27, ATCC 43895, and 472. The methods used were similar to the second study. The leaves were incubated at 4, 8, RT, and 37oC in Petri dishes with moistened filter papers. EHEC survival was determined using standard plate count on nalidixic acid containing Sorbitol MacConkey agar. No survivors were detected when treated with BEC8 or TC separately at low inoculum level after 24 h at RT on lettuce and spinach. However, when the EHEC inoculum size and/or incubation temperature increased, the efficacy of BEC8 and TC decreased. When the two treatments were combined, no survivors were detected after 10 min at all temperatures on lettuce and spinach. These results indicated that the phage cocktail and TC combination was highly effective against EHEC on leafy greens.Item Investigating the Effects of Antidepressants on Intestinal Bacteria(2024-04-16) Lebakken, Sophia; Basting, Christopher M; Bailey, Melisa; Schroeder, Ty; Broedlow, Courtney A; Guerrero, Candace; Hemmila, Charlotte; Klatt, Nichole RIntroduction: The gut-brain axis (GBA) involves bidirectional communication between the gastrointestinal tract and brain, which contains many species of bacteria that play an important role in this communication. Major depressive disorder is often treated with antidepressant medications (ADMs) that pass through the gastrointestinal tract; however, the possible adverse effects of ADMs on the gut microbiome are not well characterized. Methods: This project investigates the impact of three selective serotonin reuptake inhibitors, sertraline, fluoxetine, citalopram; one norepinephrine and dopamine reuptake inhibitor, bupropion; and one tetracyclic antidepressant, mirtazapine, on the growth of eight species of gut bacteria, Bacteroides fragilis, Bifidobacterium longum, Bacteroides uniformis, Collinsella aerofaciens, Prevotella copri, Escherichia coli, Akkermansia muciniphila, and Lactobacillus plantarum. Bacteria were treated with various concentrations of each ADM to determine potential impact on growth. We calculated the concentration of drug needed to inhibit growth by 50% (IC50) using spectrophotometry. Results Several ADMs inhibited gut bacterial growth. At 50% bacterial growth inhibition, the most prominent was sertraline (28.742 μM), followed by bupropion (43.976 μM), then fluoxetine (76.449 μM). Citalopram (244.738 μM) and mirtazapine (294.316 μM) exhibited far less inhibition. Discussion These findings suggest ADMs have antibiotic effects that disturb the microbiome resulting in potential consequences for microbiota-GBA interactions. Building on these results, future experimentation will measure uptake and metabolism of ADMs by exposing bacteria to each drug longitudinally. Metabolites will be characterized using liquid chromatography-mass spectrometry. Conclusion Given the profound impact of the gut microbiome on the gut-brain axis, these data provide novel insights into potential mechanisms by which ADMs could have unintended consequences on the gut that may perpetuate, instead of treat, mood disorders thus the microbiome should be further investigated in relation to ADMs.Item Monitoring of phenotypic and genotypic changes in antimicrobial resistance in clinical swine bacterial isolates circulating in the U.S.(2019-08) Hayer, Shivdeep SinghStarting 2017, a Veterinary Feed Directive was implemented in food animal production in the U.S. This directive prohibits the extra-label use and use of medically important antimicrobials for growth promotion. Analysis of long-term trends of antimicrobial resistance (AMR) can help in evaluating the success of such policies. The objectives of this dissertation were to monitor phenotypic and genotypic changes in antimicrobial resistance in clinical swine bacterial isolates (Escherichia coli, Streptococcus suis, Actinobacillus suis, Pasteurella multocida and Haemophilus parasuis) circulating in the U.S. between 2006 to 2016. For E. coli, the prevalence of resistance to most of the antimicrobials remained constant or changed only modestly, with the exception of enrofloxacin resistance which increased from nearly 0% in 2006 to 21% in 2016. For S. suis and P. multocida, prevalence of resistance did not change drastically except for a few antimicrobials. For A. suis and H. parasuis, statistically significant changes were estimated for several antimicrobials. However, a lack of clinical breakpoints or epidemiological cut-offs hindered in the making any clinical or epidemiological inferences. E. coli isolates resistant to ceftiofur and enrofloxacin were selected and whole genome sequencing was conducted on these isolates. Nearly 25% of the ceftiofur resistant E. coli isolates carried ESBL genes and 24% of enrofloxacin resistant isolates carried qnr genes. These genes have been reported only rarely in food animals in USA. Select plasmids carrying ESBL genes were assembled and these were similar to ESBL plasmids present globally. Additionally, these isolates were also found to be carrying mcr-9 and fosA7 genes, which have not been reported in food animals in USA previously. In addition to these studies, a systematic review on global prevalence of AMR in E. coli of swine origin is also presented. This review highlights the disparity between AMR prevalence in high income versus lower-middle income countries and a clear lack of harmonization in studies conducted worldwide.