The Chlamydiaceae are widespread pathogens of both humans and animals. Chlamydia trachomatis infection causes blinding trachoma and reproductive complications in humans. Chlamydia pneumoniae causes human respiratory tract infections and atypical pneumonia. Chlamydia suis infection is associated with conjunctivitis, diarrhea, and failure to gain weight in domestic swine. Chlamydial infections in humans and domesticated animals are generally controlled by antibiotic treatment—particularly macrolides (usually azithromycin) and tetracyclines (tetracycline and doxycycline). Tetracycline-containing feed has also been used to limit infections and promote growth in livestock populations, although its use has decreased because of growing concerns about antimicrobial resistance development. Because Sandoz and Rockey published an elegant review of chlamydial anti-microbial resistance in 2010, we will review the following: (i) antibiotic resistance in C. suis, (ii) recent evidence for acquired resistance in human chlamydial infections, and (iii) recent non-genetic mechanisms of antibiotic resistance that may contribute to treatment failure.

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Master of Science / Department of Biomedical Science / H. Morgan Scott / The modern phenomenon of increasing prevalence of antibiotic resistance in clinically relevant bacteria threatens humanity’s ability to use antibiotics to treat infection in both humans and animals. Despite the marked complexity of bacterial evolution, there is tremendous importance in unfolding the process by which antibiotic resistance genes emerge, disperse, and persist in the natural world. This thesis investigates certain aspects of this process in two experimental studies that differ primarily by scale but also by methodology. The first study examined the long-term annual prevalence of ceftiofur and tetracycline resistance in Canadian beef cattle from 2002 to 2011 at both phenotypic and genotypic levels. Ceftiofur was present at a very low prevalence (<4%) that did not statistically increase over the decade (p<0.05). Relative proportions of tetracycline genes tet(A), tet(B), and tet(C) also did not significantly change over the observation period. However, it was surprising that almost 20% of isolates recovered from nonselective agar harbored tet(C) given that current literature generally indicates that tet(C) is significantly less prevalent than tet(A) or tet(B). The usage of historical samples in addition to parallel selective plating using agar supplemented with antibiotics provided insight into systemic bias present in common microbial approaches. Long-term sample freezing significantly diminished the recoverability of E. coli over time. Additionally the usage of selective MacConkey agar containing tetracycline biased the proportions of tetracycline genes to over-represent the tet(B) gene in commensal E. coli compared to nonselective MacConkey agar. The second study attempted to explain the short-term selection effects of antibiotic treatment on the overall ecological fitness of commensal E. coli using bacterial growth parameters estimated from spectrophotometric growth curves as a simple surrogate of general fitness. Treating cattle with either tetracycline or ceftiofur was found to not only select in favor of tetracycline resistant bacteria, but also increased the overall fitness among the tetracycline resistant population. However, growth curves were unable able to explain why transiently selected resistant bacteria were eventually replaced by susceptible bacteria once the selection pressure was removed.

Autoinducer 2 (AI-2) is a quorum sensing signal employed by bacteria to coordinate their response to environmental stresses. The objective of this study was to determine the relationship between presence of AI-2 molecules, exposure to sub-therapeutic tetracycline, the expression of genes associated with the conjugal transfer of antibiotic resistance plasmids, and the conjugal transfer of these plasmids in Escherichia coli. The studies showed that AI-2 activity increased in Tets E. coli in the presence of tetracycline (2 µg/mL) under both batch and continuous culture conditions. The presence of AI-2 molecules induced tetracycline tolerance development in Tets E. coli. The studies showed that the survival rates of Tets E. coli exposed to AI-2 molecules were significantly higher compared to the cells not exposed to AI-2 molecules or cells that were exposed to only LB (Lauria-Bertani) broth. Molecular analyses using real-time PCR indicate that the expression of at least one conjugation-associated gene (trbC) is increased 9-fold in cells exposed to AI-2 molecules in the presence of sub-therapeutic tetracycline compared to its negative controls. The transconjugation frequency of the plasmid RP4 carrying the tet(A) gene increased between 10-100 fold in the presence of AI-2 molecules. In companion studies, AI-2-like activity was detected in fish, tomatoes, cantaloupes, carrots and milk samples. Interestingly, ground beef and poultry meat contained substances that appear to inhibit AI-2 activity. Collectively, these results highlight the potential importance of bacterial quorum sensing signals such as AI-2 in the response of bacterial cells to environmental stimuli and the possible role of quorum sensing signals in the quality and safety of foods.

Lo scopo di questo lavoro di tesi era di stabilire la frequenza di lattobacilli resistenti all’eritromicina e alla tetraciclina sia nel prodotto finito che nella filiera di produzione di un salame DOP del Nord Italia. Il controllo della filiera produttiva comprendeva la pelle, la carne macinata e le feci di otto suini mentre il prodotto finito era analizzato a 0, 21, 35 e 45 giorni di stagionatura. Sono state isolate colonie di lattobacilli da terreni di coltura selettivi addizionati di antibiotici. Le specie antibiotico resistenti più frequentemente isolate erano Lactobacillus sakei, Lactobacillus curvatus and Lactobacillus plantarum. I geni che con maggior frequenza sono stati evidenziati nei ceppi isolati dalla filiera e nel prodotto finito erano tet(M) ed erm(B) mentre tet(W) ed erm(B) lo erano per i ceppi isolati dalle feci. Questo studio ha consentito di fornire dati circa la presenza e la diffusione di lattobacilli tetraciclina ed eritromicina resistenti in un salame tipico del Nord Italia. I lattobacilli antibiotico resistenti potrebbero trasferire ad altri batteri, patogeni o non patogeni, i geni responsabili della resistenza stessa. Alla luce di tutto questo l’80% dei salami analizzati possono essere considerati sicuri da questo punto di vista mentre il 20% può essere valutato come un prodotto a rischio anche se la situazione non è ancora preoccupante. / The scope of this study was to assess the frequency of erythromycin and tetracycline resistant lactobacilli in the production chain (skin, minced meat, and stools of eight swine, the natural casing, and the final product at days 0, 21, 35, and 45 of ripening) of a Protected Designation of Origin dry sausage from the North of Italy and in the end products. We isolated colonies of lactobacilli from selective medium supplemented with erythromycin or tetracycline and the most frequently antibiotic resistant species isolated were Lactobacillus sakei, Lactobacillus curvatus and Lactobacillus plantarum The most frequent resistance genes in process line strains were tet(M) and erm(B) while tet(W) and erm(B) were common in strains isolated from swine stools. This study provides evidence of the presence of tetracycline and, to a lesser extent, erythromycin resistant lactobacilli in fermented dry sausages produced in Northern Italy. Although these antibiotic resistant lactobacilli could serve as reservoir organisms, in our study 80% of salami could be considered as safe even though 20% could represent a border line situation regarding the possibility of transferring antibiotic resistant genes to pathogens.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 112-144).

This master thesis is focused on the role of horizontal gene transfer in disseminating tetracycline resistance among bacteria in farm soils. In the experimental part, plasmids carrying antibiotic resistance, were exogenously isolated in biparental matings with cattle manure and Escherichia coli K-12 CV601 gfp recipients.

A case control study was performed in the conservancy area of the Kruger National Park (KNP), South Africa to find out whether the faeces of impala (Aepyceros melampus) were more likely to contain tetracycline resistant Escherichia coli (TREC) when they drank from rivers that contained these bacteria, compared to rivers that were uncontaminated with TREC. Five perennial rivers (Crocodile, Letaba, Olifants, Sabie and Sand) were selected. A total of 11 points in these rivers were sampled on three separate occasions and cultured for E. coli. Impala herds within 5 kilometres of each water collection site were identified and between 5 and 10 fresh faeces were collected for each collection period (n=209 faecal specimens). Selective culturing of E. coli was done and the resulting colonies were screened for tetracycline resistance by using the Lederberg Replica Plating (LRP) method. Resistant colonies were those that grew in the presence of 4 mg/L of tetracycline. Both a resistant and/or a susceptible isolate were then selected from each specimen, and subjected to the minimum inhibitory concentration (MIC) micro-broth dilution test for tetracyclines. The breakpoint for the MIC method was considered ≥ 8 mg/L (which is higher than for the LRP method). Twenty one of the 33 water specimens examined were found to be contaminated by E. coli. Among them (n=21), 76.19% (n=16) were resistant to tetracycline using the LRP method, although using the MIC method only 19.05% (n=4) were resistant. All of the resistant strains originated from the Letaba, Olifants and Crocodile rivers (TRECpos rivers). Among the 209 impala faeces sampled, 191 were positive for the presence of E. coli (91.38%). Within these (n=191), 36.64% (n=70) showed TREC using the RPL method, while using the MIC, 9.95% (n=19) were found to be TREC. The RPL and MIC methods showed a concordance of only 48%. Resistant isolates screened by PCR for tet(A) and tet(B) genes were found to be negative and it was concluded that other resistant genes were responsible. The odds ratios (OR) showed that impala drinking from TRECpos rivers were 19.3 (2. 63-141.68) times more likely to be infected with TREC than unexposed impala / Dissertation (MSc)--University of Pretoria, 2007. / Paraclinical Sciences / unrestricted

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