Proteus mirabilis PM13 is a well characterized chloramphenicol-sensitive isolate which spontaneously gives rise to resistant colonies on solid media containing chloramphenicol (50ug/ml) at a plating efficiency of between 10-4 and 10-5 per cell per generation. When a chloramphenicol resistant colony is grown in liquid medium in the absence of the antibiotic for I50 generations a population of predominantly sensitive cells arises. The cat gene responsible for the phenomenon is chromosomal, and has been cloned from P.mirabilis PMI3 with DNA prepared from cells grown in the absence or the presence of chloramphenicol. Recombinant plasmids which confer resistance to chloramphenicol carry an 8.5-kb PstI fragment irrespective of the source of host DNA. The location of The cat gene within the PstI fragment was determined by Southern blotting with a cat consensus 'active - site' oligonucleotide (5'-CCATCACAGACGGCATGATG-3') corresponding to the expected amino acid sequence of the active site region of chloramphenicol acetyltransferase. DNA sequence analysis has revealed a high degree of homology between the P. mirabllls cat -gene and the type I ca-t variant (Tn9), 76% at the amino acid level and 73% when nucleotides in the coding sequence are compared. The mechanism for the appearance and disappearance of chloramphenicol resistance in P. mirabilis appears to be associated with a host-specific trans-acting element which controls cat gene expression. A precedent for such a control network is given by phase variation in Salmonella typhimurium, where an invertible DNA segment controls the transcription of a trans-acting regulatory element. A comparison of the 5' regions of the S.typhimurium flagellin genes in and H2, which are alternately expressed by a flip-flop control mechanism with the 5' region of P.mirabilis cat show blocks of homology. Whether or not this homology is significant in the regulation of cat gene expression has not been determined.

Antibiotics are frequently used in aquaculture for the treatment of bacterial diseases. In this study, bacteria were isolated from the spleen, liver and kidney of grouper from PCG and cobia from EMD and THOD in southern Taiwan. All isolates were cultured on TCBS agar, blood agar, and MacConkey agar. The results showed that the isolates from PCG were 83% (20/24) Vibrio and 13% (3/24) £]-hemolysis¡F76% (22/29) Vibrio and 24% (7/29) £]-hemolysis from EMD¡F100% (6/6) Vibrio and none of £]-hemolysis from THOD. The bacteria were tested for antibiotics resistance by the disc agar diffusion method. 70% bacteria resisted to penicillin, cephazolin, and streptomycin while double resistance to furazolidone/streptomycin increased to with time from 0% to 83% in PCG. In EMD, 70% bacteria resisted to streptomycin and furazolidone, and double resistance to furazolidone/streptomycin increased with time from 0% to 60%. In THOD, 50% bacteria resisted to £]-lactam drugs, 100% bacteria resisted to cephazolin, and 67% bacteria doubly resisted to ampicillin/amoxycillin, cephalexin/cephazolin, cephalexin/streptomycin and cephazolin/streptomycin. Further, the detection of tetB, tetD, tetM, tetS and tetX resistance, tetracycline resistance genes, in the chromosomal DNAs from 17 multiple resistance isolates were performed by PCR. The PCR products were confirmed by digestion of restriction enzymes. The data indicated that J39-1, J39-2, K26-4 and K27-2, strains from THOD, together with N18-5 and M35-2, from PCG, were identified as carrying tetB. From EMD, The tetB and tetM genes were detected in P19-1, P19-3, P32-1 and Q8-3, whereas strain O2-3 carried tetS gene.

Acinetobacter baumannii is an important nosocomial pathogen, frequently associated with morbidity and mortality in immunocompromised patients due to the immuno-ablative treatments, neutropenia and prolonged hospitalization. The ability of A. baumannii to survive in the healthcare setting makes it a frequent problematic pathogen in cancer centres. Much of the interest in A. baumannii has been attributed to its remarkable rapid acquisition of resistance mechanisms A. baumannii is an excellent example of genetic plasticity, with its ability to acquire and express resistance in plasmids and chromosome particularly to carbapenems The aim of this thesis is to look at the molecular epidemiology and resistance mechanisms of 34 non-duplicate A. baumannii in two cancer centres in Cairo, Egypt. Initial sequencing of the ubiquitous blaOXA-51-like gene revealed a large diversity within the strains, with eight different genes identified: blaOXA-64, blaOXA-65, blaOXA-66, blaOXA-69, blaOXA-71, blaOXA-78, blaOXA-94, blaOXA-89/100. Typing with Pulsed-field Gel Electrophoresis (PFGE) showed an overall similarity at only 28.69% between the isolates, with variation in pattern for isolates with similar blaOXA-51-like genes. Typing with Multilocus Sequence Typing (MLST) identified 6 new Sequence Types: ST408 - ST414, in addition to ST331 and ST108 which have been previously found in other regions of the world. All three OXA-type carbapenemases: blaOXA23, blaOXA40 and blaOXA58, responsible for conferring carbapenem resistance were found in the collection studied. Insertion sequences ISAba1, ISAba2 and ISAba3 have been found to upregulate the expression of blaOXA genes. ISAba1 was found upstream of blaOXA23 in 18 strains in this collection The first report of ISAba2 was identified upstream of a blaOXA-51-like gene in this collection. Additionally, ISAba3 was bracketing the blaOXA58 genes, and two isolates harboured hybrid promoters with IS1006 and IS1008 interrupting the upstream ISAba3 sequence. Resistance to Ceftazidime was mediated by Extended-spectrum β-lactamase (ESBL) genes belonging to PER-like group: blaPER-1, blaPER-7 and the first report of blaPER-3 gene and its genetic environment in A. baumannii. In conclusion, this study shows the diversity exhibited by A. baumannii in Egypt. The various resistance mechanisms illustrate the ability of A. baumannii in acquiring and expressing resistance genes, either on plasmids or in the chromosome. Furthermore, the results indicate an urgent need to strict infection control policies and surveillance of antimicrobial use in Egyptian hospitals.

Les bactéries de l'environnement et du sol en particulier sont des producteurs actifs de molécules antibiotiques et les composés antibiotiques utilisés en médecine ont pour la plupart été isolés de bactéries saprophytes du sol qui ont elle mêmes développé une variété de mécanismes pour contrer les effets des antibiotiques conduisant à un arsenal de gènes de résistance à des antibiotiques dans l'environnement (ARGD). Une évaluation de l'abondance et de la diversité en terme de gènes de résistance à des antibiotiques a donc été conduite. Pour cette analyse, nous avons compilé 71 jeux de données de séquences d'ADN métagénomique environnementale variées: océans, et identifié des gènes de résistance pour chacun d'eux. Le sol est confirmé par cette étude comme un environnement extrêmement divers en terme de résistance à des antibiotiques. Cet étude in silico a été complété d'abord par une approche en microcosmes visant à étudier les effets soit de pollution soit par des molécules antibiotiques pures, soit par des effluents de ferme utilisés pour la fertilisation des sols. Les microcosmes de sols ont été incubés pendant 6 mois au laboratoire en conditions contrôlées. L'abondance de gènes de résistance à des antibiotiques a été évaluée au cours du temps par PCR quantitative. Une seconde étude visant à évaluer l'impact de la consommation de molécules antibiotiques par une population humaine sur son environnement immédiat, dont le sol, a été entreprise. Le village de Trois-Sauts est situé sur les berges du haut-Oyapock en Guyane Française. Les prescriptions antibiotiques sont très récentes dans cette région et les molécules distribuées ont été précisément répertoriées. Un transect de sol de 3km a été échantillonné chaque 600m afin de vérifier l'existence d'un gradient d'anthropisation entre le village (0m) et les échantillons de forêt les plus distants (3000m). Tous nos résultats confirment la présence à une forte abondance de gènes de résistance à des antibiotiques dans l'environnement, et en particulier dans le sol. Les facteurs à l'origine de la sélection et de la dissémination des gènes de résistance restent cependant difficiles à appréhender dans des environnements aussi complexes. C'est cependant avec une meilleure compréhension des phénomènes conduisant à l'émergence et à la dissémination des gènes de résistance à des antibiotiques au sein des flores pathogènes, depuis leur réservoir environnemental, que nous pourrons agir en vue de préserver les antibiotiques encore actifs aujourd'hui et ceux encore à développer. / Environmental bacteria and especially soil bacteria are active producers of antibiotic molecules and most drugs used nowadays are isolated from saprophytic soil bacteria and these microorganisms have also evolved numerous resistance pathways leading to an arsenal of Antibiotic Resistance Genes Determinants (ARGD) known as the environmental resistome. A survey of ARGD prevalence is required in order to characterize this natural phenomenon with critical implications in our current infectious diseases management. In order to perform such analysis we compiled a set of 71 metagenomic datasets from various environmental origins: soils, oceans, lakes, human feces, indoor air, etc., and compared their sequences with a database of known antibiotic resistance gene determinants (ARGD). ARGD-annotated reads are found in every environment analyzed confirming their ubiquity. Soil is found to be the richest and shares a large part of ARGD with the human gut microbiome, indicating ARGD transfers between these environments. Experiments using qPCR and metagenomic DNA sequencing on soil samples from two sites with known and distinct antibiotic pollution history were conducted to understand how ARGD abundance and diversity in soil are affected when impacted by antibiotic molecules. The first site is a reference soil from a long-term experiment without history of antibiotic pollution (Rothamsted Park Grass, UK). Soil microcosms are setup with addition of either antibiotic-containg animal manure or pure molecules and incubated for 6 months to monitor changes in ARGD concentration following these perturbations. Our second study-site is a very remote settlement in French Guiana where antibiotics are available since recently and may have impacted the local soil microbial community. Soil samples are taken following a line-transect going from the village (antibiotic source) to 3km deep in the forest in a gradient of human-impact. Our results all confirm prevalence of ARGD in soil at significant abundance but also that ARGD distribution is more correlated to environmental factors such as soil type, microbial taxonomy composition or microcosms incubation conditions than antibiotic molecules exposure in both sites. Pathogens ARGD diversity is far lower than ARGD diversity found in the environment and not all the soil resistome is readily accessible for transfer. In order to characterize the soil mobile gene pool, a strategy is proposed to isolate specifically mobile DNA directly from the environment for sequencing purposes. Better knowledge on the microbial ecology factors limiting ARGD transfers to pathogens may greatly help us reduce the current threat on our limited medical antibiotic molecules resource.

<p>To date five classes of resistance proteins have been identified in plants and these include the intracellular protein kinases, receptor-like protein kinases with extracellular leucinerich repeat (LRR) domain, LRR proteins that encode membrane bound extracellular proteins, toxin reductase and intracellular LRR proteins with a nucleotide-binding site (NBS). These proteins recognise &ldquo / invading pathogen&rdquo / and in turn trigger defence response systems that act to protect plants from invading pathogens. The NBS-LRR genes which constitutes the major class encode a family of resistance proteins that are made up of a centrally located nucleotide binding site domain and a C-terminal leucine rich repeat receptor. This class of genes constitute the largest family of resistance genes identified in plants to date. They make up the majority of proteins involved in the plant basal and inducible defence systems against pathogen infection.</p>

<p>To date five classes of resistance proteins have been identified in plants and these include the intracellular protein kinases, receptor-like protein kinases with extracellular leucinerich repeat (LRR) domain, LRR proteins that encode membrane bound extracellular proteins, toxin reductase and intracellular LRR proteins with a nucleotide-binding site (NBS). These proteins recognise &ldquo / invading pathogen&rdquo / and in turn trigger defence response systems that act to protect plants from invading pathogens. The NBS-LRR genes which constitutes the major class encode a family of resistance proteins that are made up of a centrally located nucleotide binding site domain and a C-terminal leucine rich repeat receptor. This class of genes constitute the largest family of resistance genes identified in plants to date. They make up the majority of proteins involved in the plant basal and inducible defence systems against pathogen infection.</p>

Thesis (M.Sc.)--University of Wollongong, 2004. / Typescript. Includes bibliographical references: leaf 90-98.

Philosophiae Doctor - PhD / To date five classes of resistance proteins have been identified in plants and these include the intracellular protein kinases, receptor-like protein kinases with extracellular leucinerich repeat (LRR) domain, LRR proteins that encode membrane bound extracellular proteins, toxin reductase and intracellular LRR proteins with a nucleotide-binding site (NBS). These proteins recognise "invading pathogen" and in turn trigger defence response systems that act to protect plants from invading pathogens. The NBS-LRR genes which constitutes the major class encode a family of resistance proteins that are made up of a centrally located nucleotide binding site domain and a C-terminal leucine rich repeat receptor. This class of genes constitute the largest family of resistance genes identified in plants to date. They make up the majority of proteins involved in the plant basal and inducible defence systems against pathogen infection. / South Africa

Antibiotic resistance is currently a global threat spanning clinical, environmental, and geopolitical research domains. The environment is increasingly recognized as a key node in the spread of antibiotic resistance genes (ARGs), which confer antibiotic resistance to bacteria. Detecting ARGs in the environment is the first step in monitoring and controlling antibiotic resistance. In recent years, next-generation sequencing of environmental samples (metagenomic sequencing data) has become a prolific tool for the field of surveillance. Metagenomic data are nucleic acid sequences, or nucleotides, of environmental samples. Metagenomic sequencing data has been used over the years to detect and analyze ARGs. An intriguing instance of ARGs is the multi-functional ARG, where one ARG encodes two or more different antibiotic resistance functions. Multi-functional ARGs provide resistance to two or more antibiotics, thus should have evolutionary advantage over ARGs with resistance to single antibiotic. However, there is no tool readily available to detect these multi-functional ARGs in metagenomic data. In this study, we develop a computational pipeline to detect multi-functional ARGs in metagenomic data. The pipeline takes raw metagenomic data as the input and generates a list of potential multi-functional ARGs. A plot for each potential multi-functional ARG is also created, showing the location of the multi-functionalities in the sequence and the sequencing coverage level. We collected samples from three different sources: influent samples of a wastewater treatment plant, hospital wastewater samples, and reclaimed water samples, ran the pipeline, and identified 19, 57, and 8 potentially bi-functional ARGs in each source, respectively. Manual inspection of the results identified three most likely bi-functional ARGs. Interestingly, one bi-functional ARG, encoding both aminoglycoside and tetracycline resistance, appeared in all three data sets, indicating its prevalence in different environments. As the amount of antibiotics keeps increasing in the environment, multi-functional ARGs might become more and more common. The pipeline will be a useful computational tool for initial screening and identification of multi-functional ARGs in metagenomic data. / Master of Science / Antibiotics are the drug to fight against the infection of bacteria. Since the first antibiotic was discovered in 1928, many antibiotic drugs have been developed. At the same time, scientists discovered many genes responsible for the resistance of antibiotic drugs. Nowadays, antibiotic resistance is a global threat. Detecting antibiotic resistance genes in the environment is the first step toward monitoring and controlling antibiotic resistance. In recent years, next-generation sequencing has been widely used to get the DNA sequence from the environment. Metagenomics analysis has been used over the years to detect and analyze ARGs. In the literature, it has been reported that a single gene could carry two parts of sequences corresponding to two different ARGs, thus conferring resistance to two different antibiotics. This fusion might have some evolutionary advantages. In this study, we developed a novel computational tool to detect multi-functional ARGs. We collected data from three sources: the treatment plant water, the hospital wastewater, and the reclaimed water, and identified 19, 57, and 8 potential bi-functional ARGs in each source, respectively. After we manually inspected the result, we found three most likely bi-functional ARGs. We also found one bi-functional ARG that appears in all three datasets. The gene is responsible for aminoglycoside and tetracycline resistance. The tool will serve as the initial screening step to detect multi-functional ARGs.

The ubiquitous nature of antibiotic resistance and antibiotic resistance genes (ARGs) among environmental pathogens from a variety of wastewater effluents, suggests that the aquatic environment, and specifically alternative wastewater treatment systems, may act as reservoirs for drug resistant bacteria and ARGs, thereby contributing to the widespread dissemination of antibiotic resistance. More research is necessary to contribute to our understanding of the occurrence, fate, and mobility of antibiotic resistance and ARGs among bacterial indicators of faecal contamination as well as pathogenic bacteria within Canadian wastewater treatment systems. The primary objective of this research was to determine the prevalence, fate, and potential transfer of bacterial resistance and ARGs among selected environmental pathogens exposed to alternative wastewater treatment systems, while considering the impact of treatment strategies on the expression of antibiotic resistance. A detailed analysis was initially conducted with respect to the characterization and quantification of microbial populations (including antibiotic resistant bacteria) in a variety of treatment systems and waste effluent sources. Traditional culture-based screening techniques in combination with molecular characterization (through colony or multiplex PCR), and molecular quantification using real-time quantitative PCR were utilized in order to help establish a preliminary environmental assessment of selected pathogens (Escherichia coli, Enterococcus spp., Salmonella spp.) and ARGs (tetA, blaSHV, & ampC) within a variety of wastewater treatment systems (lab-scale mesocosms, constructed wetland, constructed lagoon system, and pilot-scale biological nutrient removal (BNR) system). Overall, the level of multiple antibiotic resistance (MAR) among culturable indicator (E. coli & Enterococcus spp.) and environmental bacteria was high (reaching 100% in several instances) within different types of wastewater treatment systems and effluent sources (poultry waste effluent, municipal wastewater, aquaculture wastewater). Common antibiotic resistance profiles among E. coli isolates included simultaneous resistance to between three and five antimicrobials, whereas common MAR profiles among Enterococcus spp. isolates showed resistance to ten or more antibiotics. Real time quantitative PCR was used to determine the concentration of three bacterial pathogens; E. coli, Enterococcus faecalis, and Salmonella spp., and three ARGs; tetA, ampC, and blaSHV, within a variety of wastewater samples. Based on the results, it was concluded that high concentrations of ARGs were present in the treated effluent (10⁴- 10⁶ target gene copies/100 mL), regardless of system type (i.e. constructed lagoon, pilot-scale BNR, or constructed wetland), which may ultimately serve as a potential route for entry of ARGs and antibiotic resistant bacteria into the natural environment. Water is considered an important medium for transfer of resistance genes and resistant bacteria to the broader environment. Few studies have examined the transferability via conjugation of ARGs in E. coli and Salmonella spp. isolated from wastewater. Identification of three resistance determinants (tetA, strA, strB) conferring resistance to tetracycline and streptomycin was performed on selected multi-drug resistant Salmonella spp. and E. coli isolates. The potential for transfer of tetracycline and streptomycin resistance genes was demonstrated through broth conjugation experiments using multi-drug resistant Salmonella spp. and E. coli isolates as donors, and E. coli K12 as the recipient. Conjugation was successfully observed in 75% (9/12) of donor isolates, occurring in both Salmonella spp. and E. coli isolates. Six strains (50%) were capable of transferring their tetA, strA, and strB genes to the recipient strain, resulting in 58.5% (38/65) of total transconjugant strains acquiring all three resistance determinants. The results confirm the role of environmental bacteria (isolated from wastewater treatment utilities) as a reservoir of antibiotic resistance and ARGs, containing mobile genetic elements, which are capable of disseminating and transferring ARGs. As concerns about water quality and environmental contamination by human and agricultural effluents have increased, it has become increasingly more important to consider the prevalence and transferability of ARGs to opportunistic and human pathogens. As observed in this research, the ubiquitous nature of multi-drug resistant bacteria in water and wastewater effluents, the presence of diverse ARGs of human and veterinary health significance, as well as the transfer of resistance determinants through conjugative plasmids to recipient bacteria, suggests that environmental exposure through contact or consumption with contaminated water is probable. However, a lack of critical information still exists regarding the movement of resistance genes within and between microbial populations in the environment. In addition, the extent of human exposure to ARGs and antibiotic resistant bacteria is still not well understood, and future studies on human exposure to these resistant contaminants are necessary.