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.

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.

Our battle against pathogens has become a challenge due to the rise in antibiotic resistance and the dwindling number of new antibiotics entering the clinic. Most antibiotics owe their origins to soil bacteria, which have been producing these natural products for millennia. The rifamycins are products of actinomycetes and semisynthetic derivatives of these have been very successful in the clinic. Rifampin (RIF) has been a cornerstone agent against tuberculosis for over 50 years. In the clinic, pathogens typically develop RIF resistance by mutation of the drug. Nonetheless, a number of diverse RIF resistance mechanisms have been described, including enzymatic inactivation. Environmental bacteria are multidrug resistant, likely due to sharing the same niche as antibiotic producers and represent a reservoir of ancient resistance determinants. Furthermore, these resistance determinants have been linked to pathogens. Exploring the antibiotic resistome, the collection of all antibiotic resistance determinants from the global microbiota, reveals the diversity and evolution of resistance and provides insight on vulnerabilities of our current antibiotics. Herein, I describe a diverse collection of RIF-inactivating mechanisms from soil actinomycetes. I identified heretofore unknown RIF glycosyltransferase and RIF phosphotransferase genes (rgt and rph, respectively). RGT and RPH enzymes display broad rifamycin specificity and contribute to high-level resistance. Interestingly, RIF-sensitive Gram-positive pathogens are carriers of RPH, highlighting the existence of a ‘silent’ resistome in clinically relevant bacteria and emphasize the importance of studying resistance from environmental bacteria. Furthermore, I identified a conserved upstream DNA motif associated with RIF-inactivating genes from actinomycetes and demonstrate its role in RIF-responsive gene regulation. Finally, I explore the use of a RIF-resistance guided approach to identify novel rifamycin producing bacteria. This study expands the rifamycin resistome, provides evidence of vulnerabilities of our current arsenal of rifamycin antibiotics, and offers a strategy to identify new members of this family natural product family. / Thesis / Doctor of Science (PhD)

Antibiotics are one of the most important advances in medical science, but today, antibiotic-resistant bacteria threaten this legacy. We risk losing our ability to treat acute infections, perform invasive surgeries, and exploit immunosuppressive therapies like transplantation and cancer chemotherapy. The antibiotics we use today have ancient roots and have been produced by microbial denizens of the soil for millions of years before we adopted them in the 20th century. This history has modern consequences, as strategies to resist these compounds have evolved in concert for millions of years. The result is a vast reservoir of antimicrobial resistance that exists in environmental bacteria, which have the potential to be mobilized into human pathogens and cripple our antibiotic arsenal. Here, I set out to deepen our understanding of the environmental resistome, focusing on the rifamycin antibiotics. These compounds inhibit bacterial RNA polymerase and are frontline agents for treating tuberculosis. Environmental bacteria from the phylum Actinobacteria induce the production of resistance enzymes in response to these compounds. Although mechanistic questions remain, we demonstrate that this induction stems from the inhibition of RNA polymerase by rifamycins. The induction process is known to require a specific DNA motif; here, I identify additional sequences as part of this motif and use this information to map inducible rifamycin resistance across the entire phylum. The most common rifamycin-inducible gene was an uncharacterized family of proteins annotated as DNA helicases. I investigated these proteins and discovered that they bind to RNA polymerase and displace rifamycin antibiotics, a novel mechanism of rifamycin resistance. Lastly, we repurposed this inducible system to develop an assay to screen for novel RNA polymerase inhibitors. From this screen, we identified a rifamycin immune to a common environmental resistance enzyme and a new family of rifamycin antibiotics. / Thesis / Doctor of Philosophy (PhD) / Our antibiotic arsenal consists primarily of metabolites produced by soil microbes, which humanity repurposed into life-saving medicines in the 20th century. As a direct result of the natural origin of antibiotics, resistant bacteria exist in these same environments, independent of human use. Individual genetic determinants from this reservoir can emerge in pathogenic bacteria without warning and render antibiotics ineffective. The aim of this work was to understand how environmental bacteria resist the rifamycin class of antibiotics. Firstly, I investigated the ability of some bacteria to sense the presence of rifamycins, and in response produce proteins to protect themselves. I discovered that this process requires specific DNA sequences nearby resistance genes. Using this DNA sequence as a guide I cataloged resistance genes in thousands of bacterial genomes and discovered a new mechanism of rifamycin resistance. Lastly, I exploited this rifamycin sensing system to discover new antibiotics from soil microbes.

Yes / During January 15th – 17th, 2017 a group of scientists met, under the auspices of the UK-India Centre for Advanced Technology for Minimizing Indiscriminate Use of Antibiotics, to discuss the further developments and potential solutions to antimicrobial resistance. This was the third work shop under this funding stream held in Hyderabad. The presentations and outcomes of the workshop are released here. Key out comes included the need to address improved treatment and detection of TB, delivery of antimicrobial peptides, potential strategies for combating beta-lactam resistance. / Medical Research Council

The discovery of antibiotics and their subsequent clinical use has had a tremendous and beneficial impact on human health. The β-lactam antibiotics, which include penicillins, cephalosporins, carbapenems, and monobactams, constitute over half of the global antibiotic market. However, like all antibiotics, the β-lactams are susceptible to bacterial antibiotic resistance. One of the most disconcerting manifestations of bacterial resistance to β-lactam antibiotics is the evolution and dissemination of β-lactamases, enzymes able to chemically inactivate β-lactam antibiotics. These resistance determinants are the key contributing factor to extensively-drug resistant Gram-negative pathogens, for which we are already bereft of chemotherapeutic treatment options in some cases. The coadministration of a β-lactamase inhibitor (BLI) with a β-lactam antibiotic is a proven therapeutic strategy to counter β-lactamase expression. Unfortunately, the emergence of both serine β-lactamases (SBLs) that are resistant to BLIs and metallo-β-lactamases (MBLs), which are intrinsically resistant to BLIs due to a discrete mechanism of β-lactam hydrolysis, threaten the efficacy of combination therapy. Notwithstanding this bacterial adaptation, the discovery and development of novel BLIs is an attractive strategy to evade resistance, as evidenced by the recent clinical approval of the diazabicyclooctane (DBO) SBL inhibitor, avibactam. Herein, I describe efforts directed at understanding the mechanism of avibactam SBL inhibition. Furthermore, DBO derivatives are shown to display bifunctional properties in inhibiting both β-lactamases and the targets of β-lactam antibiotics, the penicillin-binding proteins. In addition to understanding the enzymology and chemical biology of DBOs, I describe two screening campaigns directed towards discovering inhibitors of MBLs, an unmet clinical need. Using target and cell-based screening of both synthetic and natural product chemical libraries, a fungal natural product inhibitor of clinically relevant MBLs was discovered and characterized. This study expands our understanding of the mechanisms by which DBOs can be used to combat extensively drug-resistant Gram-negative pathogens. It also describes the discovery of a new natural product MBL inhibitor using a workflow that should be amenable to other resistance determinants. It’s hoped that these studies can contribute meaningfully to countering antibiotic resistance observed in clinical settings. / Thesis / Doctor of Philosophy (PhD) / Beta-lactam antibiotics like penicillin are a mainstay for treatment of bacterial infections. Bacterial resistance to these antibiotics threatens their utility and therefore new strategies are required to counter this phenomenon. Herein I describe efforts aimed at understanding new drugs and candidate drugs that act by inhibiting the function of enzymes produced by bacteria that are able to degrade beta-lactam antibiotics. Through the discovery of new molecules and an understanding of their chemical mechanism of inhibition it is believed that bacterial resistance to beta-lactam antibiotics can be reversed.