The rifamycins are a class of antibiotics which were once used almost exclusively to treat tuberculosis, but are currently receiving renewed interest. Resistance to rifamycins is most commonly attributed to mutations in the drug target, RNA polymerase. Yet environmental isolates are also able to enzymatically inactivate rifamycins in a number of ways. Recently, rifamycin resistance determinants from the environment were found to be closely associated with a so called rifamycin associated element (RAE). The region containing the RAE from an environmental strain was shown to induce gene expression in the presence of rifamycins, hinting at an inducible system for rifamycin resistance. In this work, we examine the RAE from a model organism for Streptomyces genetics, Streptomyces venezuelae. We confirm that the promoter region containing the RAE upstream of a rifamycin monooxygenase rox is inducible by rifamycins. The strains of S. venezuelae generated in this work can be used in future genetic studies on the RAE. As well, the rifamycin monooxygenase Rox was purified for the first time and characterized biochemically. The structure of Rox was obtained with and without the substrate rifampin. Steady state kinetics for the enzyme were determined with a number of substrates, and its ability to confer resistance to rifamycins was examined. Monooxygenated rifamycin SV compound was purified and structurally characterized by NMR analysis. We proposed an aromatic hydroxylase type mechanism for Rox, in which the enzyme hydroxylates the aromatic core of the rifamycin scaffold and causes a non-enzymatic C-N bond cleavage of the macrolactam ring. This is a new mechanism of rifamycin resistance, and sheds some light on the decomposition of rifamycins mediated by monooxygenation, which is still poorly understood. / Thesis / Master of Science (MSc) / Antibiotic resistance represents a major threat to global health. Infections that were once readily treatable are no longer so due to the rise in multidrug resistant bacteria. As our arsenal of effective antibiotics is depleted, new drugs are being discovered less and less frequently. This has caused the scientific community to get creative in coming up with treatments: trying combinations of antibiotics, using antibiotics which were once considered too toxic, and repurposing antibiotics for different bacteria. Rifamycins are a class of antibiotics most commonly used in the treatment of tuberculosis. However, they are becoming more widely used as a result of antibiotic resistance. There are a number of different ways bacteria can become resistant to the harmful effects of rifamycins: by modifying the target so the drug can no longer bind to it, actively pumping the drug out of the cell, or by changing the drug in some way so it is no longer effective. Bacteria in the environment use antibiotics as a form of chemical warfare to gain an advantage over their neighbours; therefore, they have had millions of years to evolve very effective methods of antibiotic resistance. By surveying what kinds of antibiotic resistance are in the environment, we can predict what we might see one day in a medical setting. In this thesis, I have studied a protein that bacteria make to inactivate rifamycins. The rifamycin monooxygenase Rox adds an oxygen to the rifamycin scaffold; this causes spontaneous cleavage of the rifamycin backbone and changes the conformation of the drug so it can no longer bind to its target. I have also investigated the regulation of this and other genes in the bacterial strain Streptomyces venezuelae. By understanding how this process works, we can potentially design inhibitors to stop this from happening, should this method of resistance ever become clinically prevalent.

No description available.

There is significant concern that agricultural use of antimicrobials leads to spill over of antimicrobial resistance (AMR) into the general population. Animal manures contain a large and diverse reservoir of AMR genes. While poultry litter is a rich source of nitrogen, it contains an abundance of AMR genes and genetic elements associated with its dissemination. Reduce this reservoir and its potential to transmit AMR to pathogens is diminished. Methods. The ability of litter microbiota to transmit AMR was assessed using a rifampicin (Rp)-resistant Salmonella Typhimurium LT2 strain, as the recipient, and an Escherichia coli pR100, plasmid donor strain. Autoclaved poultry litter served as a negative control in plasmid transfer studies. Abundance of phenotypic AMR was assessed for litter microbiota to the antibiotics: ampicillin (Ap; 25 ï ­g/ml), chloramphenicol (Cm; 25 ï ­g/ml), streptomycin (Sm; 100 ï ­g/ml), and tetracycline (Tc; 25 ï ­g/ml). qPCR was used to estimate gene load of streptomycin-resistance and sulfonamide-resistance genes aadA and sul, respectively in the poultry litter community. AMR gene load was determined relative to total population; using 16S rRNA qPCR to estimate the total bacterial load. Different growth conditions (temperature, media) and cell densities (filter vs liquid matings) were assessed to determine conditions optimal for AMR transfer from the litter community to the recipient Salmonella strain. Results. Poultry litter contained 108 CFU/g, with Gram-negative enterics representing a minor population (< 103 CFU/g). There was high abundance of resistance to Sm (106 CFU/g) and tetracycline (107 CFU/g); and a sizeable antimicrobial resistance gene (aadA- 2.63 E+6; sul- 7.01 E+10) load in poultry litter. Autoclaving reduced viable bacterial counts by >6 log10 and the antimicrobial resistance gene load was undetectable and by 4 log10 for aadA and sul, respectively. While plasmid transfer was observed between the Salmonella recipient with R100 plasmid, no AMR Salmonella were observed in matings with live or dead, poultry litter microbiota under conditions optimal for F-plasmid transfer. Conclusions. While poultry litter microbiota contains a significant AMR gene load, potential to transmit resistance is low, under conditions commonly used to assess plasmid conjugation. Autoclaving poultry litter significantly reduces the litter resistome. Several processes (ex. composting) are currently being examined to identify one that ameliorates poultry litter. / Master of Science / There is significant concern that agricultural use of antimicrobials leads to spill over of antimicrobial resistance (AMR) into the general population. Animal manures contain a large and diverse reservoir of AMR genes. While poultry litter is a rich source of nitrogen, it contains an abundance of AMR genes and genetic elements associated with its dissemination. Reduce this reservoir and its potential to transmit AMR to pathogens is diminished. Methods. The ability of litter microbiota to transmit AMR was assessed using a rifampicin (Rp)-resistant Salmonella Typhimurium LT2 strain, as the recipient, and an Escherichia coli pR100, plasmid donor strain. Autoclaved poultry litter served as a negative control in plasmid transfer studies. Abundance of phenotypic AMR was assessed for litter microbiota to the antibiotics: ampicillin (Ap; 25 ï ­g/ml), chloramphenicol (Cm; 25 ï ­g/ml), streptomycin (Sm; 100 ï ­g/ml), and tetracycline (Tc; 25 ï ­g/ml). qPCR was used to estimate gene load of streptomycin-resistance and sulfonamide-resistance genes aadA and sul, respectively in the poultry litter community. AMR gene load was determined relative to total population; using 16S rRNA qPCR to estimate the total bacterial load. Different growth conditions (temperature, media) and cell densities (filter vs liquid matings) were assessed to determine conditions optimal for AMR transfer from the litter community to the recipient Salmonella strain. Results. Poultry litter contained 108 CFU/g, with Gram-negative enterics representing a minor population (< 103 CFU/g). There was high abundance of resistance to Sm (106 CFU/g) and tetracycline (107 CFU/g); and a sizeable antimicrobial resistance gene (aadA- 2.63 E+6; sul- 7.01 E+10) load in poultry litter. Autoclaving reduced viable bacterial counts by >6 log10 and the antimicrobial resistance gene load was undetectable and by 4 log10 for aadA and sul, respectively. While plasmid transfer was observed between the Salmonella recipient with R100 plasmid, no AMR Salmonella were observed in matings with live or dead, poultry litter microbiota under conditions optimal for F-plasmid transfer. Conclusions. While poultry litter microbiota contains a significant AMR gene load, potential to transmit resistance is low, under conditions commonly used to assess plasmid conjugation. Autoclaving poultry litter significantly reduces the litter resistome. Several processes (ex. composting) are currently being examined to identify one that ameliorates poultry litter.

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.

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.

No description available.

The MexCD-OprJ multidrug efflux pump of Pseudomonas aeruginosa confers resistance to a range of antimicrobials. Although not expressed under normal laboratory conditions, exposure to the membrane-active biocides, chlorhexidine or benzalkonium chloride, results in mexCD-oprJ expression. This suggests that membrane disruption provides the inducing signal. Consistent with this, increased mexCD-oprJ expression was demonstrated in the presence of additional membrane-damaging agents including polymyxin B, ethanol, SDS, EDTA, the organic solvents n-hexane and p-xylene, and the antimicrobial peptides melittin, V8 and V681. MexCD-OprJ expression was initially verified through increased resistance to known MexCD-OprJ antimicrobial substrates and subsequently using a mexC-lacZ transcriptional fusion and RT-PCR. Since the P. aeruginosa sigma factor AlgU is responsive to envelope stress, it was of interest to ascertain whether AlgU is capable of mediating this increased mexCD-oprJ expression. Thus, the impact of AlgU loss on mexCD-oprJ expression in response to membrane-damaging agents was assessed in a algU strain. In contrast with above, little or no mexCD-oprJ expression (assessed using resistance to MexCD-OprJ antimicrobial substrates, the mexC-lacZ transcriptional fusion and RT-PCR) occurred in response to membrane-damaging agents in the algU strain, consistent with AlgU playing a role in the envelope stress inducibility of mexCD-oprJ. Overall, envelope stress, and the ability to react to this stress through AlgU, appears to play an important role in mexCD-oprJ induction. This suggests an important role for MexCD-OprJ in alleviating envelope stress, independent of its ability to export and provide resistance to antimicrobials. A gene, PA4596, whose product shows substantial homology to the NfxB repressor of mexCD-oprJ expression, occurs downstream of mexCD-oprJ and shows AlgU-dependence and chlorhexidine inducibility, suggesting a role in the chlorhexidine-induced, AlgU-mediated expression of mexCD-oprJ. Thus, the impact of PA4596 loss on mexCD-oprJ expression was assessed. Paradoxically, the loss of PA4596 increases mexCD-oprJ expression in wild-type cells in response to chlorhexidine treatment (as assessed through RT-PCR), while its loss compromises mexCD-oprJ expression in an nfxB mutant. Nonetheless, this suggests that PA4596 is involved in the induction of mexCD-oprJ and that its ability to induce mexCD-oprJ differs depending on the state of nfxB. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2007-07-31 12:03:52.535

No description available.

Antibiotic resistance is increasing worldwide, which is threatening the effectiveness of even the most potent and recent antibiotics. The successful treatment of disease is hampered due to the multidrug resistant (MDR) phenotype exhibited by the bacterial pathogens. Therefore, the aims of this thesis were to investigate MDR through several different approaches. Integrons are important contributors to the MDR profile of nosocomial isolates within Australia, therefore the incidence of integrons was assessed in a collection of 72 conjugative clinical plasmids isolated from E. coli, a cohort of 30 urinary tract infection (UTI) isolates and a cohort of four bacteria producing metallo-beta-lactamases (MBLs). Integrons were found in 63% (45/72) of the conjugative plasmids by polymerase chain reaction (PCR). Sequencing of gene cassette arrays revealed that cassettes of the dfr and aadA families were most common. Within the cohort of UTI bacteria, 37% (11/30) were positive for class 1 integrons, and the dfrA17-aadA5 gene cassette array was most common. The four MBL-producers contained the gene cassette blaIMP-4 found within a class 1 integron which was responsible for the MBL phenotype. An assay based on real-time PCR was also developed to measure the recombination activity of the integron integrase (IntI) enzymes. The existing method of IntI measurement, the in vivo conduction assay, was used as a basis for the development of the real-time PCR assay. Five 59-be from the gene cassettes aadB, orfA, sat2, dfrA1 and aacA4 were cloned as recognition sites used in the real-time PCR assay. IntI1 was the most active integrase and showed an activity of 2.31 ?? 10??-1 when recombining the aadB and orfA 59-be. The highest level of class 2 integrase activity was 2.00 ?? 10-1?? during recombination of the sat2 and dfrA1 59-be, while IntI3 showed its highest recombination frequency of 2.29 ?? 10-1?? when the aadB and orfA 59-be were used. Additionally, the real-time PCR assay was used assess the levels of IntI activity over time. Using this method, the level of recombination as time progressed remained stable at a level of 4.10 ?? 10-2????. MDR was also analysed in 37 Acinetobacter baumannii isolates which were collected from four hospitals in Sydney. Minimum inhibitory concentration (MIC) analysis to 25 antibiotics revealed that all isolates showed a reduced susceptibility to between five and 24 antibiotics. PCR was performed to detect the presence of resistance determinants. Class 1 integrons encoding resistance to aminoglycosides, antiseptics and disinfectants were found in 35 % (13/37) of the isolates. Aminoglycoside resistance genes including aphA1 (12/37), strA (1/37) and strB (22/39) were also found. Resistance to beta-lactams was also observed in all isolates, which correlated with the presence of the ampC and blaOXA-51-like genes. The insertion sequence ISAba1 which provides an alternative promoter leading to increased gene expression was found upstream of the ampC gene in 29 isolates; the same isolates also contained the identical insertion sequence upstream of the carbapenemase resistance gene blaOXA-23. These 29 isolates also possessed the tetracycline resistance gene tetB. All but one of these 29 isolates also contained the gene blaTEM-1. Resistance to quinolones and fluoroquinolones was attributed to the presence of a Ser83-Leu83 gyrA mutation present in 36 resistant isolates. Furthermore, a putative dihydrofolate resistance gene, folA, was found in all isolates. Repetitive extragenic palindromic PCR revealed the presence of seven clonal groups. Overall, this study demonstrated the widespread impact and dissemination of MDR within nosocomial settings in Australia. The use of new assays, such as the real-time PCR assay developed in this thesis, is essential to the understanding of dissemination of antibiotic resistance.

Class of 2008 Abstract / Objectives: To evaluate changes in parental knowledge regarding antibiotic use and antibiotic resistance with an educational intervention given at elementary school parent-teacher association (PTA) meetings. Methods: This was an analytical pre-test/post-test study of an educational intervention given at two elementary schools in the Phoenix metro area. The primary dependent variable was a knowledge measure, calculated as a total score. The changes between the pre- and post-test total score means were compared using a dependent t-test. The a-priori alpha level used was 0.05. Results: The study sample consisted of 25 participants. Study data were collected between September 2007 and December 2007. The mean (SD) pre- and post-test scores were 33.7 (4.4) and 40.7 (2.7), respectively (p < 0.05). Conclusions: The educational intervention presented at elementary school PTA meetings resulted in a significant knowledge increase regarding the appropriate use of antibiotics when pre- and post-test scores were compared.