The rapid and wide scale environmental spread of multi-drug resistant bacteria is a seriousissue in recent years. Drug resistant bacteria have already occupied different ecologicalniches in many places, from wilderness to densely populated urban areas. To investigate theecological niches in Bangladesh samples were collected from wild migratory bird speciesOpen Bill Stork (Anastomus oscitans) and from the nearby water sources where these birdsvisited. A total of 76 E. coli isolates from the 170 OBS (Open Bill Stork) fecal samples and8 E. coli isolates from 3 river sources were isolated. Disk diffusion was used for checking thesusceptibility of the isolates against antibiotics that are common in human and veterinarymedicine in Bangladesh. It was found that 28.95%OBS and all water E. coli isolates wereresistant to at least one of the tested antibiotics. Common resistant phenotypes wereAmpicillin, Tetracycline, Aztreonam, Nalidixic Acid and Ciprofloxacin. Multi-drugresistance identified from 2.63%OBS and most of the water isolates. Very fewESBL(Extended Spectrum Beta-Lactamase) producing E. coli were found from OBS,whereas 50% of E. coli water isolates were ESBL producer, with all the ESBL producerspossessing the CTX-M-15 gene. The most concerning aspect of our findings was the presenceof human associated E. coli sequence types in water samples, for example ST156-complex156, ST10-complex10 and ST46. This study concludes the contaminationof environmental niches in Bangladesh by resistant bacteria.

Pseudomonas aeruginosa is an opportunistic pathogen found in soil and aquatic environments that possesses a broad range of intrinsic antibiotic resistance mechanisms, including a highly impermeable outer membrane and several RND-type efflux pumps that export a number of clinically relevant antibiotic classes. Chronic P. aeruginosa infections in cystic fibrosis (CF) patients gradually develop high levels of resistance to antimicrobial therapy due to conditions that favour the acquisition and selection of numerous chromosomal mutations, the nature of which are poorly understood. To identify chromosomal contributors to aminoglycoside resistance a P. aeruginosa transposon mutant library was screened for increases in aminoglycoside susceptibility. Six genes of interest (pstB, lptA, faoA, amgR, PA0392, and PA2798) were identified, the deletion of which meaningfully decreased aminoglycoside minimum inhibitory concentrations in wild-type P. aeruginosa. Combinations of gene deletions were constructed to determine if any of these genes contributed to aminoglycoside resistance via a common mechanism or whether they operated independently to promote intrinsic aminoglycoside resistance. In all cases, double deletion had an additive impact on aminoglycoside susceptibility, suggesting that each gene of interest contributes to resistance through an independent mechanism. Deletions in pstB, lptA, faoA, amgR, PA0392, and PA2798 were introduced into pan-aminoglycoside-resistant CF-lung isolates where they dramatically compromised aminoglycoside resistance, indicating that these genes also contribute to acquired aminoglycoside resistance in chronic P. aeruginosa infections. A fluorimetric assay was developed to measure aminoglycoside-induced membrane depolarization using the voltage sensitive probe DIBAC4(3). Gentamicin-induced membrane depolarization was found to be substantially increased in the amgR, pstB, and PA0392 mutant strains when compared to wild-type P. aeruginosa. These increases in depolarization paralleled declines in cell viability as measured by a gentamicin killing assay, suggesting that the cytoplasmic membranes of these mutant strains are more sensitive to the membrane perturbing effects of aminoglycoside-induced mistranslated proteins, and supporting a role for the disruption of the selective barrier of the cytoplasmic membrane in the bactericidal activity of the aminoglycosides. This study describes novel contributors to intrinsic and acquired aminoglycoside resistance in P. aeruginosa, and highlights the importance of membrane functions in resisting these activities. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2012-09-21 21:27:23.303

Inducible chomosomal AmpC β-lactamase (AmpC) is present in many Gram-negative opportunistic human pathogens. Expressed in response to β-lactam antibiotics, AmpC is an enzyme that can deactivate an extended spectrum of β-lactam antibiotics and thereby promote bacterial survival. Inducible chromosomal ampC is associated with ampR, a gene that encodes a LysR-type transcriptional regulator that suppresses ampC expression in the absence of β-lactam exposure. Together, ampR and ampC form a divergent operon with overlapping promoters to which the AmpR protein binds and regulates the transcription of both genes. AmpR induces ampC expression by interacting with 1,6-anhydro-N-acetylmuramyl peptide, an intermediate of peptidoglycan recycling that is generated by a glycoside hydrolase encoded by nagZ. Given the role of NagZ and AmpR in the AmpC induction pathway, the structure and function of these proteins were investigated to understand the molecular basis for how they participate in AmpC production. The crystal structure of NagZ from Vibrio cholerae was determined in complex with the glycoside hydrolase inhibitor PUGNAc (O-(2-Deoxy-2-N-2-ethylbutyryl-D-glucopyranosylidene)amino-N-phenylcarbamate) to 1.8 Å resolution. Since PUGNAc also inhibits functionally related human enzymes, the structure of the enzyme was also determined in complex with the NagZ selective PUGNAc derivatives N-butyryl-PUGNAc (2.3 Å resolution) and N-valeryl-PUGNAc (2.4 Å resolution). These structural studies revealed the molecular basis for how 2-N-acyl derivatives of PUGNAc selectively inhibit the bacterial enzyme NagZ. The effector binding domain of AmpR from Citrobacter Spp. was determined to 1.83 Å resolution and lead to the identification of a putative effector molecule binding site. In vivo functional analysis of site directed mutants of AmpR containing amino acid substitutions at the base of the putative binding pocket verified its role in AmpR function. A protocol was subsequently devised to purify milligram quantities of soluble full-length AmpR. Biochemical and biophysical analysis, including non-denaturing mass spectrometry and small angle X-ray scattering, revealed that the purified full-length protein is tetrameric and specifically binds ampC promoter DNA. In summary, this research provides the basis for the development of small-molecules that could specifically block the activity of these proteins to suppress AmpC β-lactamase production during β-lactam therapy.

No description available.

No description available.

The main aim of this work was to investigate the nature, distribution and transmissibility of tetracycline resistance (Tc^R) genes among ruminant anaerobic bacteria. Two Tc^R rumen strains of <I>Butyrivibrio fibrisolvens,</I> 1.230 and 1.23, were able to transfer the resistance phenotype to the type strain, 2221^R although a third Tc^R strain, 1.210, could not. PCR amplification of 16S rDNA sequences showed that the three isolates were phylogenetically distinct from the recipient strain, but related to each other. Hybridisation work suggested the presence of two chromosomal Tc^R determinants among the <I>B. fibrisolvens </I>isolates. All three strains contained a non-transferable <I>tet</I>(O) gene, 100% identical at the nucleotide level with <I>tet</I>(O) from <I>S. pneumoniae. </I>The mobile Tc^R determinant present in strains 1.230 and 1.23, proved to be a novel ribosome protection <I>tet </I>gene, <I>tet</I>(V), whose gene product shares only 68% amino acid identity with its closest relatives, TetO/TetM and has G+C content considerably higher than that of other <I>B. fibrisolvens </I>genes. <I>tet</I>(V) was also identified in two Australian rumen <I>B. fibrisolvens </I>strains, in the rumen anaerobes <I>Selenomonas ruminantium </I>and <I>Mitsuokella multiacidus, </I>and in a pig isolate of <I>M. multiacidus. </I>These results provide evidence for gene transfer between obligate and facultative anaerobes from different gut ecosystems and different geographical locations. PFGE demonstrated that mobile chromosomal elements 40-50 kb in size, Tn<I>B1230 </I>and Tn<I>B123, </I>with preferred insertion sites in the recipient genome mediated the transfer of <I>tet</I>(V) in <I>B. fibrisolvens. </I>No homology was found between Tn<I>B1230</I> and regions from Tn<I>916</I> and Tn<I>5253. </I>Tn<I>B1230</I> is not associated with <I>tet</I>(V) in the other bacterial strains, suggesting that a diverse range of elements carry the gene in different bacteria. Although <I>tet</I>(V) is chromosomally encoded in the majority of the strains examined, there is some evidence that the gene may be located in a plasmid in <I>S. ruminantium </I>FB32 and FB34.

Inducible chomosomal AmpC β-lactamase (AmpC) is present in many Gram-negative opportunistic human pathogens. Expressed in response to β-lactam antibiotics, AmpC is an enzyme that can deactivate an extended spectrum of β-lactam antibiotics and thereby promote bacterial survival. Inducible chromosomal ampC is associated with ampR, a gene that encodes a LysR-type transcriptional regulator that suppresses ampC expression in the absence of β-lactam exposure. Together, ampR and ampC form a divergent operon with overlapping promoters to which the AmpR protein binds and regulates the transcription of both genes. AmpR induces ampC expression by interacting with 1,6-anhydro-N-acetylmuramyl peptide, an intermediate of peptidoglycan recycling that is generated by a glycoside hydrolase encoded by nagZ. Given the role of NagZ and AmpR in the AmpC induction pathway, the structure and function of these proteins were investigated to understand the molecular basis for how they participate in AmpC production. The crystal structure of NagZ from Vibrio cholerae was determined in complex with the glycoside hydrolase inhibitor PUGNAc (O-(2-Deoxy-2-N-2-ethylbutyryl-D-glucopyranosylidene)amino-N-phenylcarbamate) to 1.8 Å resolution. Since PUGNAc also inhibits functionally related human enzymes, the structure of the enzyme was also determined in complex with the NagZ selective PUGNAc derivatives N-butyryl-PUGNAc (2.3 Å resolution) and N-valeryl-PUGNAc (2.4 Å resolution). These structural studies revealed the molecular basis for how 2-N-acyl derivatives of PUGNAc selectively inhibit the bacterial enzyme NagZ. The effector binding domain of AmpR from Citrobacter Spp. was determined to 1.83 Å resolution and lead to the identification of a putative effector molecule binding site. In vivo functional analysis of site directed mutants of AmpR containing amino acid substitutions at the base of the putative binding pocket verified its role in AmpR function. A protocol was subsequently devised to purify milligram quantities of soluble full-length AmpR. Biochemical and biophysical analysis, including non-denaturing mass spectrometry and small angle X-ray scattering, revealed that the purified full-length protein is tetrameric and specifically binds ampC promoter DNA. In summary, this research provides the basis for the development of small-molecules that could specifically block the activity of these proteins to suppress AmpC β-lactamase production during β-lactam therapy.

No description available.

No description available.

Daptomycin is a lipopeptide antibiotic that contains 13 amino acids and an N-terminally attached fatty acyl residue. The antibiotic kills Gram-positive bacteria by membrane depolarization. It has long been assumed that the mode of action of daptomycin involves the formation of oligomers on the bacterial cell membrane; however, at the outset of my studies, this had not been experimentally demonstrated. In the work described in this thesis, I have used fluorescence energy transfer (FRET) between native daptomycin and an NBD-labeled daptomycin derivative to demonstrate that the antibiotic indeed forms oligomers on bacterial cell membranes. In a liposome model, oligomer formation depends on calcium and on phosphatidylglycerol (PG). The oligomer forms rapidly and is stable for a length of time longer than required for the bactericidal effect. Through variation of the ratio of FRET donor (native daptomycin) and acceptor (NBD-daptomycin), I have determined that the oligomer consists of approximately 6–7 molecules, or, depending on the structure of the oligomer, possibly up to twice that number. Oligomer formation on liposomes and on bacterial membranes was confirmed using excimer fluorescence of a perylene-labeled daptomycin derivative. Excimer fluorescence was also used to demonstrate a stoichiometric interaction between daptomycin and PG. It has previously been shown that the bactericidal activity of daptomycin requires calcium and correlates with the concentration of PG in the bacterial cell membrane; these requirements mirror those observed here for oligomer formation. Furthermore, membrane permeabilization is selective, and electron microscopy of bacterial membranes exposed to daptomycin has revealed no discontinuities or accretions of electron density. Both of these findings suggest formation of a small membrane lesion, which is compatible with the small size of the oligomer that was determined here. In conjunction with these previous findings, the experiments contained in my thesis strongly suggest that the oligomer is the bactericidal form of daptomycin.