Expression of the mexCD-oprJ multidrug efflux operon is enhanced by the presence of membrane damaging agents [e.g., the biocide chlorhexidine (Chx)] or mutations in the nfxB gene encoding a repressor of efflux gene expression, both dependent on the AlgU envelope stress response sigma factor. Details of mexCD-oprJ regulation are, however, lacking. In examining the mexCD-oprJ locus, a gene, PA4596, encoding a homologue of NfxB (61% identity) was identified downstream of oprJ, a location conserved in all sequenced Pseudomonas aeruginosa isolates and in Pseudomonas putida. Opposite to mexCD-oprJ, PA4596 expression was reduced by Chx exposure, as assessed using RT-PCR; although like mexCD-oprJ, this was AlgU-dependent (i.e., lost in a ΔalgU strain). Deletion of PA4596 had no impact on Chx resistance indicating that it is not required for Chx-inducible mexCD-oprJ expression/ MexCD-OprJ-dependent Chx resistance. In contrast, mexCD-oprJ expression and the attendant multidrug resistance of nfxB deletion mutants were compromised upon deletion of PA4596, indicating that PA4596 plays a positive role in mexCD-oprJ expression in such mutants. Consistent with this, PA4596 expression increased in nfxB deletion and missense mutants in parallel with mexCD-oprJ. Intriguingly, mexCD-oprJ expression and multidrug resistance were observed in a mutant lacking an nfxB mutation (demonstrating an NfxB-like phenotype) and in an nfxB missense mutant and these were not compromised upon deletion of PA4596. Thus, mexCD-oprJ hyperexpression can be both PA4596-dependent and -independent. A bacterial 2-hybrid assay revealed a PA4596-PA4596 interaction, consistent with the protein forming dimers as NfxB has been shown to do. Two-hybrid assays also demonstrated that NfxB and PA4596 interact. While the functional significance of this remains to be elucidated, it is consistent with their common role in regulating mexCD-oprJ expression and is suggestive of a complex and possibly novel regulatory mechanism. These data highlight the complexity of mexCD-oprJ regulation and the apparently multiple pathways to efflux gene expression, suggestive of multiple roles for this efflux system in P. aeruginosa independent of antimicrobial efflux. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2009-08-18 14:25:18.107

Pseudomonas aeruginosa is a common soil organism as well as an opportunistic human pathogen. Treatment of P. aeruginosa infections is often complicated by innate resistance to a variety of antimicrobials mediated by multidrug efflux systems. The MexAB-OprM efflux system is constitutively expressed in wildtype strains and contributes to innate antimicrobial resistance, while hyperexpression of the system results in acquired high levels of resistance. MexAB-OprM is hyperexpressed in nalC mutants containing mutations in the gene encoding NalC, a repressor of a two-gene operon, PA3720-armR. armR encodes a protein modulator of MexR, a repressor of mexAB-oprM expression. Previous reports showed that genes encoding the MexAB-OprM efflux system are upregulated in response to pentachlorophenol (PCP), a phenolic compound that is a common environmental contaminant. Induction of mexAB-oprM and PA3720-armR by PCP was confirmed using RT-PCR, and MexAB-OprM was shown to be involved in PCP resistance. An electromobility shift assay (EMSA) showed that PCP interacts with NalC, interfering with its binding to the PA3720-armR promoter region and thereby promoting PA3720-armR expression. Nonetheless, the increase in ArmR did not drive mexAB-oprM expression suggesting that PCP induction of this efflux operon occurred via a different mechanism. A direct PCP-MexR interaction could not be demonstrated using an EMSA. PCP exposure did, however, reduce expression of nalD, encoding a second repressor of mexAB-oprM, which might explain the PCP-promoted increase in mexAB-oprM expression. PCP is unlikely to be the intended inducer(s)/substrate(s) for this system but probably resembles these. Several compounds related to PCP were tested for an interaction with NalC but all were negative in EMSAs. Plants produce a variety of phenolic compounds, which are often antimicrobial and, so, root extracts of various plants were tested for an ability to interact with NalC and interfere with promoter binding. Extracts from Boehmeria tricuspis, Uncaria tomentosa and Ixiolirion tataricum were shown to interact with NalC, suggesting that plant compounds may be the intended inducers/substrates for NalC/MexAB-OprM. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2010-09-10 10:35:16.271

Magister Pharmaceuticae - Mpharm / The epidemiology of drugs is 'defined as the manner in which drugs are used by doctors, nurses, pharmacists and patients. As such epidemiology of antimicrobial drugs is the manner in which this class of drugs is used by healthcare professional, patients or caregivers. Antimicrobial drugs have a pivotal role worldwide in preventing infections and treating infectious diseases. The challenge that lies in the health sector is to maintain antimicrobials' effectiveness by using them appropriately to avoid toxicity, adverse reactions and resistance among other problems. The world faces a future in which ten million people could die annually due to infections that are resistant to available antibiotics. Despite people already dying of drug-resistant infections in private and public hospitals, doctors are still prescribing antibiotics for viral infections, for which they have no effect. The aim of this research is to study the epidemiology of antimicrobial drugs and factors that lead to the inappropriate use of antimicrobials, which is resulting in a steep rise of antimicrobial resistance in the private sector from a community pharmacy perspective.

This dissertation documents the development of an environmental framework for monitoring antimicrobial resistance gene (ARG) dissemination in the aquatic environment. The work opens with a review of the relevant literature and outlines the importance of an environmental framework for monitoring ARG dissemination as part of antimicrobial resistance risk assessments. The ability to interrogate sequencing data quickly and easily for the presence of ARGs is crucial in order to facilitate their monitoring in the environment. As current laboratory methods for the detection and surveillance of antimicrobial resistant bacteria in the environment were limited in their effectiveness and scope, the dissertation begins by describing the design and implementation of a Search Engine for Antimicrobial Resistance (SEAR), a pipeline and web interface for detection of horizontally-acquired ARGs in raw sequencing data. The suitability of metagenomic methods for monitoring the ARG content of effluents from faecal sources was then assessed via a pilot study of a river catchment. Novel metagenomes generated from effluents entering the catchment were interrogated for ARGs. The relative abundance of ARGs in effluents were determined to be higher relative to the background environment, as were sequences relating to human and animal pathogens and mobile genetic elements. Thus, effluents were implicated in the dissemination of ARGs throughout the aquatic environment. To determine if ARGs were potentially in use in the environment, the expression of ARGs within effluents was then evaluated across a series of longitudinal samples through the use of metatranscriptomics, and the presence of potential environmental antimicrobial selection pressures was examined. This demonstrated that the abundance of ARGs, as well as antimicrobial usage at the effluent source, was correlated with the transcription of ARGs in aquatic environments. The work described in this dissertation has also found that horizontally transmitted ARGs were present in pathogenic endospore-forming bacteria commonly found across the aquatic environment, potentially providing a mechanism for ARG persistence in the environment. Finally, these findings were integrated into a universal framework for monitoring ARG dissemination in aquatic environments and used to highlight the developments required to incorporate this framework into future environmental ARG research and to facilitate antimicrobial resistance risk assessments.

Antimicrobial resistance is a serious and imminent threat to human health, though its rise may be controlled with improved stewardship strategies that limit the emergence and spread of resistant strains. Motivated by theoretical models from population genetics and ecology, my M.Sc. experimentally evaluates how varying drug availability in either time or space impacts the prevalence of resistance in a population. By experimentally evolving Pseudomonas aeruginosa under different antibiotic selection regimes in vitro, I show that spatial, but not temporal, drug free refuges delay the fixation of resistance by promoting the coexistence of sensitive and resistant genotypes. Second, I establish that this polymorphism is underlain by a trade-off between resistance and growth rate in the absence of antibiotic that underpins the maintenance of diversity through negative frequency dependent selection. Third, I demonstrate that spatially varied drug selection cannot prevent the fixation of resistance because continued selection leads to the evolution of resistant types that pay smaller costs of resistance and gradually displace sensitive strains. These results provide insight into the fate of diversity under long-term selection and highlight the value of incorporating the principles of evolutionary ecology into antimicrobial resistance stewardship.

This study aims to evaluate the removal efficiency of microbial contaminants from two hospitals on-site Wastewater Treatment Plants (WWTPs) in Saudi Arabia. Hospital wastewaters often go untreated in Saudi Arabia as in many devolving countries, where no specific regulations are imposed regarding hospital wastewater treatment. The current guidelines are placed to ensure a safe treated wastewater quality, however, they do not regulate for pathogenic bacteria and emerging contaminants. Results from this study have detected pathogenic bacterial genera and antibiotic resistant bacteria in the sampled hospitals wastewater. And although the treatment process of one of the hospitals was able to meet current quality guidelines, the other hospital treatment process failed to meet these guidelines and disgorge of its wastewater might be cause for concern. In order to estimate the risk to the public health and the impact of discharging the treated effluent to the public sewage, a comprehensive investigation is needed that will facilitate and guide suggestions for more detailed guidelines and monitoring.

Introduction: Carbapenems and colistin are the last-line antibiotics for treating Gram-negative bacterial infections. However, increasing reports of resistance to these antibiotics is being reported in clinical settings, the environment and in animals. In this paper, we describe the molecular epidemiology and resistance mechanisms of colistin and carbapenem resistance in clinical, veterinary, and environmental Enterobacterales isolates in Pretoria, South Africa. Method: One hundred VITEK®-2-confirmed colistin and carbapenem-resistant clinical isolates were collected from the departmental isolate bank at the National Health Laboratory Service. A total of 88 porcine (stool) and 11 environmental (effluents) samples were collected in November 2018 and again in March 2019 from a farm in Pretoria. Both the porcine and environmental samples were screened using Eosin methylene blue agar with colistin and ertapenem disks. All isolates were identified and a minimum inhibitory concentration of colistin and carbapenems was determined using the MicroScan® WalkAway system. Isolates resistant to colistin were confirmed by the broth microdilution method. Isolates phenotypically resistant to colistin and carbapenems were selected for whole genome sequencing to determine the resistome and phylogenetic trees were drawn to determine the relatedness of isolates. Results: A total of 275 Gram-negative isolates were identified from the clinical (100), environmental (57) and veterinary (118) samples using the MicroScan® WalkAway system. The MicroScan® WalkAway system’s minimum inhibitory concentration results for clinical isolates revealed 88% and 93% resistance to colistin and carbapenems, respectively. BMD was found to be more reliable in all isolates, and it recorded higher MICs (increased resistance) than the MicroScan® WalkAway system. Overall, colistin susceptibility was higher among animal isolates compared to the clinical and environmental samples. Genomic analysis identified several resistance genes associated with resistance among the isolates and the CTX-M family were the dominant resistance genes. Phylogenomic analysis demonstrated closer evolutionary relationship between EB008 (environment), SW10B (animals), and C080 and C084 (both humans) strains as well as with strains from the United States of America, Canada, China, Russia and Durban (South Africa). Conclusion: The study established multiple resistance genes from different antibiotics to mediate resistance in Enterobacterales isolates from humans, animals and the environment. The presence of carbapenemases in animals is alarming and poses a public health concern. Strains EB008 (environment), SW10B (animals) and C080 and C084 (both human) were phylogenetically related with strains from the United States of America, China and Durban (South Africa) more commonly. Therefore, One Health approach studies are significant to ascertain colistin and carbapenem transmission from human to animals/the environment and vice versa to combat increasing resistance in Enterobacterales. / Dissertation (MSc)--University of Pretoria, 2020. / National Research Foundation (NRF) / National Health Laboratory Service research grant / Medical Microbiology / MSc / Unrestricted

archives@tulane.edu / Gram-negative bacteria secrete outer membrane vesicles (OMVs) that play critical roles in intraspecies, interspecies, and bacteria-environment interactions. Some OMVs, such as those produced by Pseudomonas aeruginosa, have previously been shown to possess antimicrobial activity against competitor species. In the current work, we demonstrate that OMVs from Burkholderia thailandensis inhibit the growth of drug-sensitive and drug-resistant bacteria and fungi and exhibit antibiofilm activity against methicillin-resistant S. aureus (MRSA) and Streptococcus mutans. We show that a number of compounds, including peptidoglycan hydrolases, 4-hydroxy-3-methyl-2-(2-non-enyl)-quinoline (HMNQ) and long-chain rhamnolipid present in B. thailandensis OMVs exert antimicrobial activity. Furthermore, we demonstrate that HMNQ and rhamnolipid possess antimicrobial and antibiofilm properties against various microbes. Rhamnolipid is superior at reducing the integrity of biofilms while HMNQ displays greater bactericidal activity. We attempted to use HMNQ and rhamnolipid to combat MRSA and promote wound healing in a murine full-thickness wound model. However, further optimization of the model and characterization of the molecules in antimicrobial efficacy, wound healing, and host immune responses are required. Overall, this work indicates that B. thailandensis secretes antimicrobial OMVs that may impart a survival advantage by eliminating competition. In addition, bacterial OMVs may represent an untapped resource of novel therapeutics effective against biofilm-forming and multidrug-resistant organisms. / 1 / Yihui Wang

Antimicrobial-resistant bacteria are a major source of ailment in modern-day nosocomial settings, with numerous risks including leading to possible further drug resistance or spreading to those who cannot fight off the infection due to immune suppression or dysfunction. Previous work in our laboratory has determined that Klebsiella pneumoniae possesses inhibitory effects on the growth of a variety of bacteria that contain antimicrobial-resistant properties in the Enterobacteriaceae family, a major source of nosocomial antimicrobial-resistance. This novel property of K. pneumoniae inhibits the growth of Citrobacter freundii, Enterobacter aerogenes, and Enterobacter cloacae through an anti-microbial protein. The antimicrobial protein secreted from K. pneumoniae has been shown to reduce the density and growth of C. freundii, E. aerogenes, and E. cloacae in both biofilm and planktonic forms. The work performed in this thesis has shown that the antimicrobial protein is plasmid mediated by introducing a transposon (Tn5) to the plasmid to provide resistant selection and a possible way to create a mutant knockout to find the exact location of the gene in the plasmid. Upon transposon mutagenesis of the plasmid, it was electroporated into Rec- E. coli. The E. coli were then able to produce the antimicrobial protein allowing the formation of zones of inhibition when screened on C. freundii, E. aerogenes, and E. cloacae lawns. Upon confirmation that the plasmid mediates the anti- microbial protein, the plasmid was sent for sequencing to further characterize the gene responsible for coding the anti-microbial protein. This novel antimicrobial protein has high sequence similarity to bacteriocins and, thus far, is a novel and uncharacterized protein of plasmid origin found in only in this particular strain of K. pneumoniae. Further research involving this new bacteriocin could aid in the development of treatments for the highly drug resistant Enterobacteriaceae family members.

The emergence and rapid spread of antimicrobial resistance (AMR) is a phenomenon that extends beyond clinical settings. AMR has been detected in multiple environmental compartments, including agricultural soils and water bodies impacted by wastewater discharges. The purpose of this research project was to evaluate what factors could influence the environmental persistence of antibiotic resistance genes (ARGs), as well as to identify potential strategies employed by human pathogens to survive in secondary environment outside the host. The first part of this dissertation describes the incidence of the New Delhi metallobeta lactamase gene (blaNDM-1) – an ARG conferring resistance to last resort antibiotics – in the influent of a wastewater treatment facility processing municipal wastewater from Jeddah, Saudi Arabia. Detection of blaNDM-1 was followed by the isolation of a multi-drug resistant strain of E. coli (denoted as strain PI7) at a frequency of ca. 3 x 104 CFU/m3 in the untreated municipal wastewater. Subsequently, we described the decay kinetics of E. coli PI7 in microcosm experiments simulating biological treatment units of wastewater treatment plants. We identified that transition to dormancy is the main strategy prolonging the persistence of E. coli PI7 in the microcosm experiments. Additionally, we observed slower decay of E. coli PI7 and prolonged stability of extracellular DNA in anoxic/anaerobic conditions. In the last chapter of this thesis, the fate of extracellular DNA is further explored. Using as a model Acinetobacter baylyi ADP1, we describe the stimulation of natural transformation frequencies in the presence of chlorination disinfection byproducts (DBPs). Moreover, we demonstrate the ability of BAA to stimulate transformation is associated with its capacity to cause DNA damage via oxidative stress. Overall, this dissertation addresses important knowledge gaps in our current understanding of ARB and extracellular ARG persistence in the environment. The results from this project highlight the importance of retrofitting the existing water treatment process with advance membrane filtration units, and the need to relook into the current disinfection strategies. Wastewater treatment technologies should be assessed for their efficacies in not only inactivating ARB and ARGs, but also whether unintended consequences such as stimulated horizontal gene transfer would occur.