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Conventionally, in Escherichia coli, phylogenetic groups A and B1 are associated with commensal strains while B2 and D are associated with extraintestinal strains. The aim of this study was to evaluate diarrheagenic (DEC) and commensal E. coli phylogeny and its association with antibiotic resistance and clinical characteristics of the diarrheal episode. Phylogenetic groups and antibiotic resistance of 369 E. coli strains (commensal strains and DEC from children with or without diarrhea) isolated from Peruvian children <1 year of age were determined by a Clermont triplex PCR and Kirby-Bauer method, respectively. The distribution of the 369 E. coli strains among the 4 phylogenetic groups was A (40%), D (31%), B1 (21%), and B2 (8%). DEC-control strains were more associated with group A while DEC-diarrhea strains were more associated with group D (𝑃 < 0.05). There was a tendency (𝑃 = 0.06) for higher proportion of persistent diarrhea (≥14 days) among severe groups (B2 and D) in comparison with nonsevere groups (A and B1). Strains belonging to group D presented significantly higher percentages of multidrug resistance than the rest of the groups (𝑃 > 0.01). In summary, DEC-diarrhea strains were more associated with group D than strains from healthy controls.

Flavomycin is a phosphoglycolipid antibiotic, which is used exclusively as a growth- promoting feed additive. Existing data, from both in vitro and in vivo ruminal studies, give conflicting results regarding its mode of action, as well as no clear microbiological basis for the observed responses. Studies however do indicate that the principal site of action of the antibiotic is the rumen. From the available data, flavomycin appears to promote growth in a manner distinct from that of other feed antibiotics, for which the growth-promoting mechanisms have been elucidated. This study aimed to characterise the effect that flavomycin has on the microflora of the rumen, allowing its growth promoting mechanism in ruminants to be determined. In vitro analysis demonstrated that flavomycin has only antibacterial activity, as ruminal species of protozoa, fungi and archaea were unaffected by the antibiotic. Of the ruminal bacterial species tested, Fusobacterium necrophorum, Fibrobacter spp. and certain hyper-ammonia producing (HAP) bacteria (Atopobium oviles, Desulfomonas sp. and Peptostreptococcus anaerobius) were highly sensitive to the antibiotic. The sensitivity of the Fibrobacter spp. to flavomycin suggested that flavomycin is likely to select for a cellulolytic bacterial flora comprised predominantly of Ruminococcus spp., as has been previously proposed on the basis of in vitro fermentation studies. In vivo, suppression of ruminal numbers of F. necrophorum and flavomycin sensitive HAP bacteria occurred as a result of flavomycin supplementation. It was demonstrated that these bacterial populations were highly variable, between individual sheep and days respectively, suggesting why previous studies produced conflicting results. Assessment of ruminal fermentation parameters demonstrated that flavomycin caused a significant decrease in the production of ruminal ammonia, which could be directly attributed to decreased numbers of ruminal HAP bacteria. A small increase in the ruminal concentration of lactate also occurred, which con-elated with the suppression of ruminal numbers of the lactate utilising F. necrophorum. No change in the balance of individual volatile fatty acids (VFA) occurred, however total VFA production was significantly decreased. This was likely to be due to the total viable anaerobic bacterial counts being lower during flavomycin supplementation, although this result was not statistically significant. Uncultured rumen bacteria were also implicated in the growth promoting mechanism of flavomycin. Molecular investigation of the rumen bacterial population by denaturing gradient gel electrophoresis (DGGE) demonstrated that several changes occurred, which correlated with flavomycin supplementation. Analysis of the sequence data obtained from excised DGGE bands highlighted that the majority of the operational taxonomic units (OTU) detected were represented by presently uncultured species of bacteria, of which almost half had not been previously identified. Identification of the flavomycin sensitive bacterial populations was not possible, however, due to the recovery of multiple sequences from individual DGGE bands. Existing bacterial 16S rDNA sequence data, from published ruminal clone libraries, also demonstrated the poor cultural representation of rumen bacterial diversity, with only 10% of the OTU detected being represented by cultured bacterial species. Based on these results, flavomycin has the ability to increase the efficiency of dietary protein utilisation, although the role of uncultured bacteria in the growth promoting mechanism of the antibiotic is not clear. Protein retention in the rumen is increased as a consequence of decreased deamination by ruminal HAP bacteria. F. necrophorum has the ability to attach to and damage rumen epithelium, as well as being the principal aetiological agent in the development of liver abscesses. Suppression of F. necrophorum is likely to decrease metabolic and immune burdens within the animal, as well as potentially reducing the rate of rumen wall tissue turnover. The use of flavomycin as a feed additive is to be banned in Europe in 2005. However, it is not known if presently available feed additives or treatments will be able to act as an effective replacement for this antibiotic. Characterisation of an adaptive resistance mechanism against flavomycin, in the ruminal bacterium Prevotella bryantii, demonstrated that cross-resistance to therapeutic antibiotics can occur. As a result of this finding, and the interest in development of phosphoglycolipid antibiotics for therapeutic application, it can be concluded that the withdrawal of the use of a flavomycin as a feed additive is a wise precautionary measure to ensure the long-term efficacy of this class of antibiotics.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Antibiotic-resistant bacterial strains continually arise and their increasing prevalence poses significant clinical and societal challenges. Functional analyses of resistant mutants and the study of general stress responses perturbed by antibiotic treatment have yielded valuable insights into how resistance arises through mutations. However, less is known about the population dynamics and communal interactions that underlie the development of resistance through mutations. In this work, we utilize systems approaches to study the functional dynamics of bacterial populations evolving antibiotic resistance. We follow a continuous culture of Escherichia coli facing increasing levels of antibiotic and show that the vast majority of isolates are less resistant than the population as a whole. We find that the few highly resistant mutants improve the survival of the populations less resistant constituents, in part, by producing indole, a signaling molecule generated by actively growing and unstressed cells. We show, through transcriptional profiling, that indole serves to turn on drug efflux pumps and oxidative stress protective mechanisms. The indole production comes at a fitness cost to the highly resistant isolates, and wholegenome sequencing reveals that this bacterial altruism is enabled by drug-resistance mutations unrelated to indole production. This work establishes a population-based resistance mechanism constituting a form of kin selection whereby a small number of resistant mutants can, at some cost to themselves, provide protection to other more vulnerable cells, enhancing the survival capacity of the overall population in stressful environments. Deeper studies into cooperative strategies bacteria use to evade antibiotics may prove critical for the rational design of more effective antimicrobial interventions. / 2031-01-01