不良研究所

2024 Annual AMR Symposium Poster presentations (alphabetical by author)

Mellouk Abdelkader,听INRS-AFSB,听UQAM,听Supervisor:听Charles Calmettes
"TAM complex and outer membrane biogenesis"

Fathima Afsal, Civil Engineering, 不良研究所, Supervisor:听Dominic Frigon
"Spatial differences in the human gut microbiota selects distinct antimicrobial resistance genes in a simulated in vitro reactor"

Paula Armoa Ortiz,听Biology,听INRS Armand Frappier, Supervisor: Charles Calmettes
"Structural and functional characterization of a putative effector protein of helicobacter pylori"

Erick听Arroyo Perez,听Microbiology and Immunology,听Universit茅 de Montr茅al,听Supervisor: Yves Brun
"Machine learning prediction of Antibiotics from growth kinetic"

惭补谤颈别尘听颁丑补濒产颈,听Microbiology,听INRS-AFSB,听Supervisor: Charles Calmettes
"Identification and characterization of new factors for the gastric adaptation in Helicobacter pylori"

Farhan Rahman Chowdhury,听Biology, Concordia University,听Supervisor: Brandon Findlay听[Flash Talk #1]
"Recycling Antibiotics: Restoring Antibiotic Susceptibility via Drug Cycling"

尝补耻谤别苍肠别听颁辞耻迟耻谤别,听Biology,听Universit茅 de Sherbrooke,听Supervisor: Jean-Philippe C么t茅
"High-throughput tools to probe Salmonella-gut microbiota dynamics"

惭补谤驳辞迟听顿别蝉蝉补谤迟颈苍别,听Biology,听Universit茅 de Sherbrooke, Supervisor: Jean-Philippe C么t茅
"Type 1 fimbriae-mediated collective protection against type 6 secretion system attacks"

Emmanuel听Diaz Mendoza,听Civil Engineering, 不良研究所, Supervisor: Dominic Frigon
"Impacts of simple versus complex wastewater substrate compositions on the fate of antimicrobial resistance genes in activated sludge wastewater treatment"

Laura Dom铆nguez Mercado,听Chemistry & Biochemistry,听Concordia University,听Supervisor: Brandon Findlay听[Flash Talk #12]
"Resistance to "evolution-proof" Oct-TriA1 occurs through mutations in phospholipid transport, outer membrane assembly, and LPS biosynthesis in E. coli"

搁辞尘补颈苍听顿耻谤补苍诲,听Institut de Biologie Int茅grative et des Syst猫mes (IBIS),听Universit茅 Laval,听Supervisor: Christian Landry
"Mutational landscape of echinocandin resistance"

贰蝉锄迟别谤听贵补谤办补蝉,听Microbiology and Immunology,听不良研究所,听Supervisor: Dao Nguyen
"Production of antimicrobials by Canadian High Arctic bacteria"

滨蝉尘补颈濒听贵濒颈蝉蝉,听Food science,听Universit茅 Laval
"Bacteriocins vs antibiotics: resistance, co-resistance and cross resistance"

Parham听Ghasemloo Gheidari,听School of Computer Science,听不良研究所,听Supervisor: J茅r么me Waldisp眉hl听[Flash Talk #11]
"Borderlands Science: Bringing together millions of players for human health!"

Claire Gibson, Civil Engineering, 不良研究所, Supervisor: Dominic Frigon
"Identifying Antimicrobial Resistance Gene Sequence Variants to Track the Dissemination of Antimicrobial Resistance Between Environmental Reservoirs"

惭补迟丑颈别耻听骋颈驳耻猫谤别,听D茅partement de Biochimie, Microbiologie et Bio-informatique, Universit茅 Laval,听Supervisor: Christian Landry [Flash Talk #7]
"Can We Predict Antifungal Resistance?"

础谤颈补苍苍补听骋颈耻谤濒别辞,听Pharmacology & Therapeutics,听不良研究所, Supervisor: Bastien Castagner
"Confronting AMR: A Comprehensive Approach through Public Awareness and Scientific Engagement"

厂辞辫丑颈补听骋辞濒诲尘补苍,听Microbiology & Immunology,听不良研究所, Supervisor: Dao Nguyen
"Anti-Psl/ PcrV monoclonal antibody potentiates in vitro neutrophil opsonophagocytic killing and in vivo bacterial clearance of Pseudomonas aeruginosa isolates from Cystic Fibrosis children that failed tobramycin eradication therapy."

翱肠茅补苍别听骋辞苍肠补濒惫别蝉,听Biology, Universit茅 de Sherbrooke, Supervisor: Jean-Philippe C么t茅听[Flash Talk #9]
"New insights into the antimicrobial resistance of Pseudomonas aeruginosa through functional genomics in the clinical context of cystic fibrosis"

顿补尘颈迟丑补听骋耻苍补迟丑颈濒补办别,听Biochemistry, Microbiology and Bio-informatics,听Universit茅 Laval,听Supervisor: Sylvain Moineau听[Flash Talk #4]
"Salmonella enterica Kentucky strains from East Afrika show high resistance to antibiotics and phage infections"

Mark Hemmings,听Biochemistry, 不良研究所, Supervisor: Albert Berghuis听[Flash Talk #6]
"Structural Studies of Non-Canonical Aminoglycoside Binding Modes"

础濒别虫补苍诲谤补听贬耻驳丑别蝉-骋辞测别迟迟别,听Biochemistry, 不良研究所, Supervisor: Martin Schmeing
"Structural Characterization of Single Module Nonribosomal Peptide Synthetase IndC"

Adarsh Jay,听Biochemistry,听Universite Laval,听Supervisor: Christian R. Landry
"Mapping copy number variation and loss of heterozygosity on antifungal resistance genes"

顿辞苍驳测耻苍听闯耻苍驳,听Food Science and Agricultural Chemistry,听不良研究所, Supervisor: Jennifer Ronholm [Flash Talk #3]听
"Utilization of conjugative CRISPR-Cas9 system for targeted elimination of cat and blaCMY-2-bearing plasmid in Escherichia coli from bovine gastrointestinal tract"

贰尘尘补听碍别濒濒测,听Biochemistry, 不良研究所, Supervisor: Albert Berghuis
"Fungal mannan cell wall synthesis proteins: A structural investigation"

尝颈补尘听碍别辞驳丑,听Pharmacology & Therapeutics, 不良研究所, Supervisor: Bastien Castagner
"Evaluating a novel non-antibiotic treatment for Clostridioides difficile infection"

础丑尘补诲听碍别锄锄辞,听Microbiology and Biotechnology, INRS, Supervisor: Salim Timo Islam
"Mechanism of synthesis and secretion of group IV E. coli capsular polysaccharide"

Abdallah Khaled,听Food sciences,听Universit茅 Laval,听Supervisor: Ismail Fliss
"Towards a new era in the fight against Campylobacter spp. : Antimicrobial peptides, a promising avenue"

Helena Leal,听Microbiology and Immunology,听Universit茅 de Montr茅al,听Supervisor: Caroline Quach
"A tale of two cities: the status of AMR spread in the water cycle of two major Canadian cities"

脡尘颈濒别听尝茅迟辞耻谤苍别补耻,听Biology, Universit茅 de Sherbrooke, Supervisor: Jean-Philippe C么t茅
"High-throughput screening: a valuable tool for identifying essential genes associated with Pseudomonas aeruginosa physiology and virulence."

贬补颈箩颈补辞听尝颈苍,听Animal Science,听不良研究所,听Supervisor: Xin Zhao听[Flash Talk #13]
"A Combined in Silico Approach Predicts Great Potentials for Horizontal Transfer of Antimicrobial Resistance and Virulence Genes from Salmonella Plasmids"

闯颈苍虫颈苍听尝颈耻,听Food Science and Agricultural Chemistry; Institute of Parasitology & 不良研究所 Centre for Viral Diseases,听不良研究所, Supervisor: Xiaonan Lu & Qian Liu听[Flash Talk #8]
"AIoT-enhanced Global Antimicrobial Resistance Automated Surveillance System Using Multiplex Microfluidic Technique"

颁测谤颈别濒濒别听惭补谤迟颈苍颈,听INRS Armand Frappier, Supervisor: Charles Calmettes
"Unraveling the crystal structure of the HpaA adhesin: Insights into cell adhesion"

骋别辞谤驳颈听惭别谤丑颈,听Quantitative Life Sciences,听不良研究所,听Supervisor: Jesse Shapiro听[Flash Talk #2]
"Understanding the role of horizontal gene transfer in the evolution of Acinetobacter"

贰尘尘补听惭耻濒丑辞濒濒补苍诲,听Microbiologie, infectiologie et immunologie,听Universit茅 de Montr茅al,听Supervisor: Yves Brun
"Identification and characterization of gene products with novel roles in outer membrane biology"

叠谤颈诲驳别迟听翱'叠谤颈别苍,听Food Science & Agricultural Chemistry,听不良研究所, Supervisor: Jennifer Ronholm听[Flash Talk #5]
"Subtle Genomic Differences in Klebsiella pneumoniae May Indicate Adaptation to Distinct Hosts"

闯补肠辩耻别濒测苍听搁颈肠丑,听Microbiology and Immunology,听不良研究所, Supervisor: Dao Nguyen
"Proteomic profiling of the conserved stringent response in stationary phase Pseudomonas aeruginosa"

Mohamed Elfateh Salem,听Animal Science,听不良研究所, Supervisor:听Xin Zhao
"Measuring Anaerobe Clostridium Viability in High Throughput"

Ori Solomon,听Microbiology and Immunology,听不良研究所, Supervisor: Marcel Behr听[Flash Talk #10]
"Fluroquinolone resistance in multidrug-resistant Mycobacterium tuberculosis strains in Vietnam"

痴补丑颈诲别丑听痴补丑诲补迟尘补苍别蝉丑, Microbiology and Immunology, 不良研究所, Supervisor: Marcel Behr
"Bacteriocins as Effective Natural Antimicrobials Against Multidrug-Resistant Clostridium Species"

A Combined in Silico Approach Predicts Great Potentials for Horizontal Transfer of Antimicrobial Resistance and Virulence Genes from Salmonella Plasmids
Objectives: Antimicrobial resistance genes (ARGs) and virulence genes (VGs) have been widely reported in non-typhoidal Salmonella (NTS) serovars which are major foodborne pathogens from poultry. This study detected replicon typing, conjugality of ARGs- and VGs-carrying plasmids from NTS of poultry origin using in-silico methods. Methods: Both PlasmidFinder2.1 and VRprofile2 were employed to detect poultry-origin NTS plasmids from the NCBI RefSeq database, and then oriTfinder was used to determine the conjugality of plasmids. The ARGs and VGs on plasmids were identified by both VRprofile2 and oriTfinder. The resistance phenotypes were predicted by ResFinder4.1 and oriTfinder, while the virulence phenotypes were predicted by oriTfinder and VRprofile2. Results: We identified 183 plasmids from 309 downloaded sequences. Among them, 77 (42.08%) plasmids were conjugative, 25 (13.67%) mobilizable, and 81 (44.26%) non-mobilizable. Fifty-one plasmids (27.9%) contained multi-replicons. One hundred five plasmids carried 58 ARGs, belonging to 11 classes. In addition, 49 plasmids carried 36 different VGs belonging to 12 virulence groups. Conclusions: The detected high rate of conjugative plasmids and existence of many multiple replicons suggest possible high rates of plasmid-mediated horizontal gene transfer (HGT) events. Detection of previously unreported plasmid-borne ARGs (blaLAP-2, mph(A), and qnrS1) and VGs (fdeC and htpB) from NTS in poultry calls for more vigilant monitoring. Back to听Haijiao鈥�鈥嬧赌嬧赌嬧赌嬧赌嬧赌�

A tale of two cities: the status of AMR spread in the water cycle of two major Canadian cities
Background and objectives. Wastewater surveillance is a promising tool for monitoring AMR in the community. Conventional wastewater treatment plants (WWTP) are not specifically designed to limit the spread of AMR. In fact, they can act as hotspots for genetic transfer, favoring resistance dissemination. In parallel, few studies have attempted to understand how human behaviours may impact the presence of AMR markers in sewage. The study aimed to identify and quantify the AMR load through the urban water cycle and survey the population to identify critical perceptions and habits that can foster AMR. Method: Samples were collected through the urban water cycle: source water, drinking water treatment plant, hospital water inlet, hospital wastewater effluent, wastewater collector system, WWTP influent, effluent, and sludge. A total of 16 sampling points were targeted, repeated over 3 separate sampling campaigns. Twenty-three antimicrobial resistance genes (ARGs) were quantified by qPCR (Resistomap). In parallel, a survey aiming to verify perceptions and lifestyle habits that may foster or prevent AMR transmission was distributed in selected neighborhoods. Results: Preliminary results suggest the detection of selected AMR genes in more than 50% of wastewater samples. As expected, an important reduction of the bacterial load was measured between wastewater influent and effluent. Analysis of the first results from the survey indicated a lack of knowledge of AMR, leading to behaviors that can promote increased resistance. For example, 28% of participants indicated using antibiotics without a prescription. Implications: A better understanding of ARGs in WWTPs and treatment effectiveness is particularly important as many WWTP infrastructures need to be upgraded. The results of the work will identify lifestyle habits and behaviors that can contribute to the spread of AMR and, therefore, provide decision-makers with evidence to guide policy action and target effective interventions to address AMR. Back to Helena

AIoT-enhanced Global Antimicrobial Resistance Automated Surveillance System Using Multiplex Microfluidic Technique
To address the spread of antimicrobial resistance (AMR) in the farm-to-fork continuum, we previously developed a real-time surveillance system to monitor the rise of AMR bacteria in the food chain using microfluidic "lab-on-a-chip" for antibiotic susceptibility testing and image recognition. However, this surveillance system only monitors one antimicrobial each time and requires costly GPU that limits its applications in agri-foods. To address this challenge, an embedded AIoT device Orange Pi, priced under $150, was used to control a homemade portable incubator and run an object detection model for identifying chromogenic reactions on multiplex microfluidic chips, which achieved a processing speed exceeding 30 fps and 99% precision in classifying bacterial growth or inhibition. The collected data could be uploaded to the cloud for analysis of large-scale AMR trends. This affordable and compact AI node holds the potential to revolutionize AMR detection across the global food supply chain. Back to听Jinxin

Anti-Psl/ PcrV monoclonal antibody potentiates in vitro neutrophil opsonophagocytic killing and in vivo bacterial clearance of Pseudomonas aeruginosa isolates from Cystic Fibrosis children that failed tobramycin eradication therapy.
New-onset Pseudomonas aeruginosa (PA) infections in Cystic Fibrosis patients are often treated with inhaled tobramycin (TOB), but eradication therapy fails in up to 40% of cases. Novel therapeutic strategies are thus needed. This study examined a bispecific monoclonal antibody (mAb) targeting Psl and the type III secretion system protein PcrV to enhance neutrophil (N蠁) mediated opsonophagocytic killing (OPK) in vitro and improve PA clearance in vivo. The mAb was tested for its ability to enhance N蠁 OPK of persistent PA isolates in vitro. An in vivo PA pulmonary infection model assessed the mAb's effect alone or with TOB on bacterial clearance. Results showed the mAb enhanced in vitro N蠁 OPK of persistent isolates and that a prophylactic dose significantly improved in vivo bacterial clearance. Preliminary studies also suggest that combining the mAb with TOB may further enhance bacterial clearance compared to single modality therapy. Back to Sophia

Bacteriocins as Effective Natural Antimicrobials Against Multidrug-Resistant Clostridium Species
Clostridium bacteria cause serious intestinal infections in humans and animals, often treated with antibiotics. However, antibiotic resistance is increasing, necessitating alternative therapies. Bacteriocins, natural antimicrobial peptides produced by bacteria, present a promising solution by selectively targeting harmful pathogens while preserving beneficial microbiota. This study evaluates the antimicrobial activity of nisin, enterocin, and brevibacillin against thirty Clostridium species. These species, characterized for metabolic activity, antibiotic resistance, and pathogenicity, exhibited diverse resistance profiles, with many being multidrug-resistant. Sensitivity to bacteriocins was assessed using minimum inhibitory concentrations (MIC), minimum bactericidal concentrations (MBC), and agar diffusion assays. Results showed that all Clostridium species were inhibited by the bacteriocins, with MIC values between 12.5 and <0.19 碌g/ml. This study highlights significant antibiotic resistance in Clostridium and demonstrates the potent antibacterial properties of bacteriocins, suggesting their potential as effective natural antimicrobials for controlling multidrug-resistant Clostridium in both human and animal applications. Back to听Vahideh

Bacteriocins vs antibiotics: resistance, co-resistance and cross resistance
Bacteriocins are natural peptides produced by several bacterial strains and having antimicrobial activity against bacteria that are genetically close to the producing strain. Recently, bacteriocins have been proposed as one of the most promising alternatives to antibiotics for promoting growth or for combating multidrug-resistant bacteria in animal production.听In this study, we used multidisciplinary approach combining microbiology, genomics and functional proteomics for the study and understanding the phenomena of co-resistance and cross-resistance between different classes of bacteriocins and between bacteriocins and antibiotics.听Our results showed that most of studied bacteriocins exhibit significant inhibition activity against multidrug resistance bacteria. No significant co resistance was observed between bacteriocins belong to different classes. Moreover, no cross resistance was detected between bacteriocin and antibiotics. This new scientific data and will contribute to the efficient, targeted and user-friendly use of bacteriocins in the animal sector.听Back to Ismail

Borderlands Science: Bringing together millions of players for human health!
Antimicrobial resistance (AMR) is a critical global health challenge that requires to mobilize all available resources. Citizen science leverages the computational capacities of individuals en masse. We created Borderlands Science (BLS), a minigame within the popular game Borderlands 3, targeting the task of aligning 1 million 16S ribosomal RNA sequences from human microbiome studies. Since its launch in April 2020, over 4 million players have completed 135 million science puzzles. Our analysis shows BLS enhances microbial phylogeny estimations and UniFrac effect sizes compared to state-of-the-art computational methods. By training an AI agent on player solutions, we plan to automate applying the human-provided alignment strategies to new data. We hope to facilitate AMR detection mechanisms by refining alignments and enhancing phylogenies, increasing the likelihood of detecting phylogenetic and taxonomic signals in applications, such as Disbyosis, which is known to be a risk factor for Clostridium difficile (C. diff) infections. Back to Parham

Can We Predict Antifungal Resistance?
Patients with generalized fungal infections face a high mortality rate. Caspofungin, an antifungal drug, specifically inhibits FKS proteins which are an essential component of the fungal cell wall. Mutations in the FKS gene sequences that confer resistance are often located in specific regions hence referred to as hotspots. This project aims to develop a tool that predicts antifungal drug resistance using the amino acid sequence of these hotspots. Single-mutation hotspot sequences and multiple-mutations hotspot sequences were inserted in S. cerevisiae. By applying a selective pressure and analyzing changes in sequence frequency, we determined resistance scores for each variant. A random forest classifier was trained on the resistance scores and amino acid properties of single-mutation hotspot sequences. When shown multiple-mutations hotspot sequences, this model classifies them in the correct category with 92% accuracy. SHAP values analysis highlights that specific amino acid properties and the mutation鈥檚 position contribute most to the model.听Back to Mathieu

Confronting AMR: A Comprehensive Approach through Public Awareness and Scientific Engagement
The antimicrobial resistance (AMR) crisis requires a comprehensive approach extending beyond the mere scientific development of novel antimicrobials. The 不良研究所 AMR Centre Student Outreach Team (SOT) addresses the lack of public awareness on this topic. The SOT aims to cultivate a deeper understanding of the consequences of AMR by actively engaging, educating, and inspiring students and the public, encouraging their involvement in the fight against AMR. Student and public outreach events are disseminated through newsletters, social media posts, and student engagement activities. We share informative, relatable, and easily digestible subject matter tailored to resonate with diverse audiences. Our social media platforms have experienced a 35% growth and the target for student participation was surpassed by 70%, reaching students from diverse departments across the university. Addressing AMR requires the integration of both scientific and social dimensions. The AMRC student outreach team bridges the gap between the public and scientific communities. Back to Arianna

Evaluating a novel non-antibiotic treatment for Clostridioides difficile infection
Clostridioides difficile is classified as one of the most urgent drug resistant pathogens by the CDC, indicating a need for novel therapeutics. Pathogenesis is mediated by the absorption of homologous toxins TcdA and TcdB into epithelial cells from the colon lumen. A thiosulfate-containing analogue of IP6, the endogenous co-factor for toxin activation, has shown efficacy in treating C. difficile infection (CDI) in a mouse model by inducing premature toxin activation in the colon lumen. Surprisingly, IP6 itself also promoted survival, despite being strongly chelated in the colon lumen and thus being incapable of inducing premature toxin activation. We hypothesize that IP6 and thiosulfate analogues work via different mechanisms in vivo. We show that IP6 inhibits the outgrowth of dormant spores into toxin-producing vegetative cells, whereas thiosulfate analogues induce more toxin activation. We are currently developing a CDI model to further examine the efficacy of these molecules. Back to Liam

Fluroquinolone resistance in multidrug-resistant Mycobacterium tuberculosis strains in Vietnam
Historically, tuberculosis treatment consisted of tuberculosis-specific antibiotics, separating antibiotic resistance pressure on Mycobacterium tuberculosis from non-mycobacterial infections. The rise of drug resistance and a trickling drug development pipeline led to implementation of broad-spectrum antibiotics such as fluoroquinolones in tuberculosis care. However, the widespread use in other infections, both microbiologically confirmed and empirically diagnosed, puts into question the sustainability of fluoroquinolone-based tuberculosis therapy. We used whole genome sequencing to identify resistance to fluoroquinolones in multidrug-resistant Mycobacterium tuberculosis strains from a clinical trial in Vietnam. Fluroquinolone resistance was geographically spread, indicating no clear epidemiological signal. On phylogenetic analysis, fluoroquinolone resistance-associated mutations were widely distributed across diverse strains. Additionally, phylogenetically-related isolates possessed different resistance-associated mutations. Together these findings suggest that fluroquinolone acquisition is happening in multiple sites, independently. Frequent resistance acquisition may have serious implications in national policies for treatment and prevention of MDR-TB.听Back to Ori

Fungal mannan cell wall synthesis proteins: A structural investigation
Invasive fungal infections are amongst some of the most deadly and difficult to treat diseases. Nevertheless, antifungal research is an understudied field, culminating in a narrow arsenal of cheaply available and non-toxic fungi-specific drugs. This has left our currently available treatment strategies alarmingly vulnerable to the rising global threat of antifungal resistance; particularly with the emergence of multidrug resistant 鈥渟uperbug鈥�, Candida auris. As such, there is a pressing need for new fungi-specific drug targets. As a feature lacking in mammalian cells, fungal cell wall glycosyltransferase proteins provide an exciting fungi-specific targeting option. Och1, a Golgi transmembrane mannosyltransferase, initiates cell wall synthesis by transferring a single 伪-1,6-mannose residue to an N-glycan core. Here, the first x-ray crystal structure of Och1 is reported, solved to 2.0 脜, providing an experimental scaffold for future structure-guided drug design of fungi-specific mannosyl-inhibitors.听Back to Emma

High-throughput screening: a valuable tool for identifying essential genes associated with Pseudomonas aeruginosa physiology and virulence
The alarming increase in infections caused by the ESKAPE pathogens, a group containing highly virulent and multidrug-resistant microorganisms, underscores the pressing need for a better understanding of them. Among them, Pseudomonas aeruginosa, an opportunistic pathogen, presents various pathogenicity factors that we have yet to fully comprehend. To deepen our understanding of P. aeruginosa infection mechanisms, high-throughput screenings have been conducted using a collection of mutants of this pathogen under conditions that best mimic the in vivo environment. This approach enables the rapid identification of new essential genes for various virulence factors, which play a crucial role in the development of infections by this pathogen. Clarifying the underlying mechanisms and their correlation with the phenotype could pave the way for the use of these genes as potential therapeutic targets.听Back to 脡mile

High-throughput tools to probe Salmonella-gut microbiota dynamics
Salmonella enterica serovar Typhimurium is a foodborne pathogen of importance as well as a model organism for the study of microbial interactions within a context of infection. By employing libraries of single-gene deletions or S. Typhimurium promoters, we can comprehensively investigate the numerous interactions that may occur between the pathogen and microbiota commensals in a large-scale manner. For example, we found that S. Typhimurium becomes tolerant to azithromycin when co-cultured with Lacticaseibacillus rhamnosus or Limosilactobacillus reuteri. Acidified media from cell-free supernatant of lactobacilli for instance, also induced the tolerant phenotype. Using a collection of single-gene deletion mutants of S. Typhimurium (SGD), we were able to determine that in presence of the lactobacilli, the pathogen undergoes changes in its metabolism that lead to increased tolerance against the antibiotic that prevails the modification in envelope homeostasis and membrane permeability observed with acidified media. Further characterization of the mechanisms underlying these interactions holds promise in enhancing our comprehension of bacterial dynamics in a context of infection and may be a great asset to the discovery of new drug targets.听Back to Laurence

Identification and characterization of gene products with novel roles in outer membrane biology
The outer membrane (OM) of Gram-negative bacteria reduces the efficacy of many antibiotics. To identify novel gene products involved in OM biology in Escherichia coli, we are screening a whole genome CRISPRi library first with the fluorescent reporter N-phenyl-1-napthylamine (NPN), then with targeted microscopy screens. As expected, initial tests show that cells with known OM gene knockdowns exhibit higher NPN fluorescence compared to controls. For microscopy, preliminary experiments demonstrate that multiple fluorescent proteins can effectively track phenotypic changes between treatments that affect different cellular pathways. We are also using machine learning models to analyze and cluster the microscopy images; control tests show that our machine learning model can cluster images in a biologically meaningful way. We are now screening our full CRISPRi genome library with NPN and microscopy to identify genes of interest involved in the OM, which will then be further characterized with more targeted assays.听Back to Emma

Identification and characterization of new factors for the gastric adaptation in Helicobacter pylori
Helicobacter pylori (H. pylori) colonizes the gastric niche, affecting around half of the global population. Persistent colonization is linked to significant gastric pathologies like gastritis, peptic ulcers, and adenocarcinoma, primarily attributed to H. pylori's acid adaptation mechanisms. However, many proteins involved in this process remain unidentified. Our focus is on identifying and characterizing new factors crucial for H. pylori's acid adaptation, potentially serving as therapeutic targets for eradication. Sequences encoding these proteins were discovered via phylogenetic analysis, particularly focusing on two novel secreted proteins: a Tip-alpha-like-protein and a alginate-like-protein, showing no sequence homology with known proteins. Structural analysis revealed similarities to TNF-alpha-inducing-protein and an alginate-lyase-enzyme. In order to validate the predicted function of Tip-alpha like protein, we studied its binding and internalization in human gastric cells.听Back to Mariem

Identifying Antimicrobial Resistance Gene Sequence Variants to Track the Dissemination of Antimicrobial Resistance Between Environmental Reservoirs
To curtail the spread of antimicrobial resistance (AMR), effective regulatory measures must be established. Wastewater treatment plants (WWTPs) and agriculture are known hotspots of AMR, however, the movement of AMR between these environments remains unclear. Effective approaches to address this question must be inexpensive and sensitive. This study aimed to apply multiplex amplicon sequencing of antimicrobial resistance genes (ARG) to identify sequence variants characteristic of municipal wastewater and agricultural sources. Thus, providing important insights to enable the dissemination of AMR to be tracked between reservoirs.听Samples were collected across Canada from 18 WWTPs, and from cattle, swine and chicken manure. High-throughput qPCR of 216 ARGs (targeting 9 antimicrobial classes) was used to quantify selected ARGs. The ARG sequence diversity and microbial community composition based on 16S rRNA gene were determined by amplicon sequencing using the Illumina MiSeq technology.听Back to Claire

Impacts of simple versus complex wastewater substrate compositions on the fate of antimicrobial resistance genes in activated sludge wastewater treatment
Although wastewater treatment plants are propagators of Antimicrobial Resistance Genes (ARGs) in the environment, the ARG dynamics in activated sludge remain unexplained. In this study, reactors received one of two synthetic wastewaters: one simple and readily biodegradable (sugars and volatile fatty acids), and the other complex and slowly biodegradable (proteins and lipids). Upstream sewer influent solids were supplied to simulate immigration.听PCR detected 81 ARGs, and the abundance of several ARGs was significantly higher between the complex and simple feeds (9 and 17 ARGs, respectively) regardless of immigration. Immigration significantly increased the concentrations of 8 genes and decreased the concentration of 4 compared to sludges where no immigration was present, regardless of the feed.听This study suggests that complex and slowly biodegradable substrates as well as immigration enhance ARG presence and diversity. This means that a greatly polluted wastewater poses a high risk of spreading antimicrobial resistance in the environment. Back to Emmanuel

Machine learning prediction of Antibiotics from growth kinetics听
The discovery of new drugs is a challenging task; the chemical space that is theoretically possible to synthesize is impossible to screen in a lifetime. Machine learning has helped in this area, since it can be used to find patterns in the structure of effective drugs in order to predict new ones.听A few studies have employed deep learning methods in the field of antibiotic discovery, and have successfully predicted novel active compounds. These studies scored antibiotic activity using a single of growth yield at the end of the incubation to label the compounds as either active or inactive. However, the data points from the entire bacterial growth curve in the screening can provide more information on the activity of chemical compounds, their potency and even about their mode of action.听We have screened a set of molecules enriched with antibiotics for their effect on growth of E. coli. We have developed methods to incorporate the data from the growth curve in the labeling of compounds, and assessed the advantages of including this data in the model for prediction of new compounds.听Back to Erick

Mapping copy number variation and loss of heterozygosity on antifungal resistance genes
This study presents an extensive copy number variation (CNV) analysis of more than 600 Candida albicans genomes. The strains have been previously assessed for their resistance to a spectrum of antifungal agents sourced from both literature and clinical isolates. The genomes were scrutinized for various types of CNVs, including whole chromosomal and segmental aneuploidy, as well as polyploidy, which are known critical mechanisms of genetic adaptation by employing multiple complementary bioinformatic methods. By constructing a comprehensive phylogenetic tree, we also identified significant hotspots of loss of heterozygosity (LOH) across different clades to uncover correlations between copy number variations and LOH, and the observed antifungal resistance profiles. The detected CNVs and LOHs were compared with known resistance-conferring mutations listed in a new database of resistance mutations to examine how these genomic changes impact resistance genes. This comparative analysis addresses the prevalence of copy number variations and their role in antifungal resistance. Back to听Adarsh

Measuring Anaerobe Clostridium Viability in High Throughput
Viability testing for anaerobes is time-consuming and expensive. Clostridium perfringens (CP) is an anaerobic bacterium that is a common cause of toxin-mediated systemic and enteric diseases. Here, we present a rapid, economical, and reliable method for testing the viability of CP using the Geometric Viability Assay (GVA). This method is efficient and cost-effective, requiring one pipette tip per sample, and is compatible with the anaerobic jar system enabling 96 viability measurements in one jar. The method has low technical bias and a dynamic range extending over 5 orders of magnitude. Using our approach, we performed antibiotic sensitivity testing to determine the minimum concentration for complete bactericidal activity (MBC) of four CP clinical isolates against four antibiotics. We found that the MBC concentration for tetracycline was up to 8x higher than the concentration for complete growth inhibition (MIC). The results confirm the efficacy of our method in screening Clostridium species viability. Back to Mohamed

Mechanism of synthesis and secretion of group IV E. coli capsular polysaccharide
Enteropathogenic E. coli O127 (EPEC) is one of the virulent species causing infantile diarrhea, with high rate of mortality.听Given the presence of capsule (group IV) which has the same repeating units structure of O-Ag LPS. That led us to work on the mechanism of synthesis of capsular polysaccharide (CPS) and probing the in commons with O-Ag LPS. With the help of virtual processes, an interiguing results were obtained. Back to Ahmad

Mutational landscape of echinocandin resistance
Echinocandins constitute one of the major classes of antifungals. They are large semi-synthetic compounds targeting an essential component of the fungal cell wall. Specifically, they act as non-competitive inhibitors of two enzymes synthesizing 尾-glucans. These enzymes are encoded by FKS1 and FKS2, two duplicate genes that are conserved in some pathogenic fungi. Unfortunately, despite echinocandin use being recommended as first line against invasive candidiasis, more and more treatments fail due to pathogens acquiring resistance. Routine sequencing of FKS1 and FKS2 from clinical isolates has shown that resistance correlates with the presence of mutations in two small regions in each gene, which have since then been referred to as 鈥榟otspots鈥�. Surprisingly, very little is known about the actual mechanism of action of this drug class, or why certain mutations lead to resistance. In this study, we used deep-mutational scanning to evaluate the level of resistance of around 120,000 variant hotspot sequences, including all possible single and double mutants, as well as sequences that are naturally found in a diverse repertoire of fungal genomes. We show that the position at which a mutation occurs in the hotspot is crucial to confer resistance to one and/or more of the three tested compounds. We used molecular dynamics simulations coupled with molecular docking to infer the specific binding of each drug. Finally, we show how the dataset can be used to train a machine-learning model to help predict echinocandin resistance from any FKS hotspot sequence.听Back to Romain

New insights into the antimicrobial resistance of Pseudomonas aeruginosa through functional genomics in the clinical context of cystic fibrosis
impact of this bacterium. Given P. aeruginosa's substantial clinical impact in cystic fibrosis (CF), the primary aim is to discover new therapeutic approaches that account for the specific features of this disease. A major challenge in this field lies in the significant disparity between in vitro antimicrobial sensitivity profiles and effective clinical response, posing a serious threat to eradicating mixed microbial communities observed in CF patient's lungs. Despite the common use of minimum inhibitory concentration tests to guide clinical decisions, these approaches often prove ineffective in treating chronic lung infections as observed in CF. Thus, this research endeavors to propose innovative antimicrobial strategies tailored to this particular pathology, thereby offering new perspectives to enhance the management of CF patients. Back to听Oc茅ane

Production of antimicrobials by Canadian High Arctic bacteria
With a global antibiotic resistance crisis and a dry research and development pipeline for new听and effective antibiotics, researchers are exploring new avenues for identifying novel antibacterial compounds. We propose to investigate bacterial isolates from the Canadian High Arctic, an extreme environment with high microbial diversity and unique environmental听pressures. The goal of this project is to determine if Arctic isolates produce secreted compounds听hat could represent novel antibiotics. Screening of Arctic isolates from our collaborators in the听Whyte lab led to the identification of six bacterial strains with antibacterial activity against听several clinically relevant pathogens. We then characterized the antibacterial activity of organic听extracts from these isolates against relevant clinical pathogens after confirming activity against听the hyper-permeable and efflux deficient E. coli 鈭哹amB 鈭唗olC. We developed a high-throughput听bioluminescence assay using E. coli 鈭哹amB鈭唗olC to identify antibiotic activity from culture听supernatants and used this assay to optimize antibiotic production of our candidate isolate.听Following fractionation of our candidate extract using reiterative rounds of High-Performance听Liquid Chromatography coupled to Mass Spectrometry we have isolated a candidate bioactive听compound. We will determine the potency and spectrum of activity of this compound against relevant clinical pathogens. The structure of antibacterial compound(s) will then be elucidated听using Nuclear Magnetic Resonance. This work will highlight the value of the Arctic bacterial听isolates as a source for novel antimicrobials.听Back to Eszter

Proteomic profiling of the conserved stringent response in stationary phase Pseudomonas aeruginosa
The stringent response (SR) is a conserved stress response induced by accumulation of the alarmones (p)ppGpp, via synthesis from the enzymes RelA and SpoT. Induction of which drives a physiological shift away from active metabolic processes towards stress management and cellular quiescence. The Pseudomonas aeruginosa (p)ppGpp-null 螖relA 螖spoT (螖SR) mutant displays numerous abnormalities, particularly in stationary phase, including increased membrane permeability, decreased antibiotic tolerance and impaired antioxidant defenses. To further understand the cellular processes under SR control, we performed a quantitative proteomic profiling of the PAO1 螖SR mutant and its isogenic parent. We detected 366 螖SR-dependent differentially expressed proteins (DEPs) in stationary phase, many of which corroborate the phenotypes observed. Down-expression proteins that respond chemotaxis stimuli, oxidative or osmotic stress and up-expression of numerous metabolic pathways were seen. This study represents the first characterization of the SR-regulated proteome, providing insight into the processes involved in SR-mediated adaptation during stationary phase.听Back to Jacquelyn

Recycling Antibiotics: Restoring Antibiotic Susceptibility via Drug Cycling
Resistance evolution massively outpaces antibiotic discovery. Here, we investigate the feasibility of restoring susceptibility to antibiotics that previously failed due to resistance (antibiotic recycling). Prior studies to recycle antibiotics attempted to use collateral sensitivity (CS), the phenomenon where resistance to one antibiotic induces hypersensitivity to another. However, we found that despite repeated evolution of CS towards the antibiotic piperacillin (PIP) upon evolution of gentamicin (GEN) resistance, subsequent PIP exposure failed to restore GEN susceptibility. This suggests that CS may not be able to drive antibiotic resensitization. Inspired by prior research from our laboratory, we attempted to achieve resensitization by leveraging the fitness burden of resistance evolution. Treating bacteria with a third antibiotic nitrofurantoin imposed significant fitness costs, restoring GEN-sensitivity in the majority of the strains and making GEN effective against bacteria that were once GEN-resistant. Recycling antibiotics by carefully applying antibiotics in a sequence may reduce the emergence of resistance.听Back to Farhan

Resistance to "evolution-proof" Oct-TriA1 occurs through mutations in phospholipid transport, outer membrane assembly, and LPS biosynthesis in E. coli
Numerous antibiotics have been labeled as 鈥淓volution proof鈥�, following routine evolutionary experiments that failed to yield resistant mutants. An example of one such antibiotics is Oct-TriA1, which binds to lipid II in Gram-negative bacteria and disrupts the proton motive force. We describe here the first reported evolution of de novo resistance to Oct-TriA1. Using a modified version of the Soft Agar Gradient Evolution method, we evolved resistance in E. coli to 8x the MIC of the ancestral strain. Whole genome sequencing of the evolved strains and allelic exchange linked the increased resistance to mutations in genes related to phospholipid transport, outer membrane assembly, and LPS biosynthesis. These findings provide crucial insights into the resistance mechanisms of tridecaptins and underscore the urgent necessity for more rigorous testing when assessing the antimicrobial resistance evolution of new compounds.听Back to Laura

Salmonella enterica Kentucky strains from East Afrika show high resistance to antibiotics and phage infections.
Thirteen Salmonella enterica strains of the serotypes Enteritidis (6 strains), Heidelberg (3), and Kentucky (4) were isolated from poultry environments in Kenya to characterize their sensitivity to antibiotics and bacteriophages. The complete genomes (PacBio) of the 13 strains were compared to 27 strains of the same serotypes isolated in other studies from farm animals (96% swine) in East Africa (Ethiopia and Kenya).听Overall, the Kentucky strains carried the most antibiotic resistance genes (CARD, ResFinder). Kirby Bauer disk diffusion assays with the 13 Salmonella strains against 13 antibiotics from 9 classes showed that the highest average antibiotic resistance (AR) was with Kentucky (4.3 AR/strain) and Heidelberg (4) strains.听Among the 13 strains, 3 out of the 4 Kentucky strains were resistant to 22 out of 24 Salmonella phages. Genome analysis of the two infecting phages showed that they possess mechanisms to evade restriction-modification systems, suggesting the importance of phage escape strategies.听Back to Damitha

Spatial differences in the human gut microbiota selects distinct antimicrobial resistance genes in a simulated in vitro reactor听
While most environmental studies determine the level of human exposure to antimicrobial resistance genes (ARGs), true risk quantification requires the combination of the exposure with the host system response. The host response maybe influenced by the pH differences in the colonic compartments, which we aim to study using an in vitro human gut reactor.听Four 10-day runs were carried out with five completely mixed semi-continuously flowing reactors simulating digestion. Community composition was assessed by 16S rRNA gene amplicon sequencing and 75 ARGs were quantified by high-throughput qPCR array (Resistomap).听Over the 10 days of reactor operation, the total abundance of certain ARG classes increased in all colonic compartments while the abundance of certain other ARG classes decreased. Towards the end of the runs as the microbial community neared steady-state, we observed differences in ARG abundance between colonic compartments, which suggest variations in the host response in ARG dissemination.听Back to Fathima

Structural and functional characterization of a putative effector protein of helicobacter pylori听
Helicobacter pylori (H. pylori) is a gram-negative bacillus, which colonizes the gastric mucosa and is capable of establishing chronic infection (gastrointestinal infections, peptic ulcer and gastric cancer). The first step in its pathogenicity is the adhesion to gastric epithelial cells. We have identified several genes present in gastric strains of H. pylori that have not yet been characterized. A putative adhesin of H. pylori related to eukaryotic fibronectin proteins is encoded by one of these genes. H. pylori could have hijacked this protein with the aim of using it in the host-pathogen interaction. To study the function of this putative adhesin we will first solve its structure by x-ray crystallography. In cellulo studies with monocytes and gastric cells will be carried out to study its effect on the regulation of gene expression, cellular localization and its possible interaction partners.听Back to Paula听

Structural Characterization of Single Module Nonribosomal Peptide Synthetase IndC
Nonribosomal peptides (NRPs) are a major group of natural product compounds, well known for their biological activities. They are clinically used for many therapeutic applications, acting as antivirals (cyclosporin A), immunosuppressants (rapamycin), antifungals (caspofungin), anticancer agents (dactinomycin), and antibiotics (penicillin, vancomycin, and daptomycin), along with having several industrial applications, including use as polymers and dye. Indigoidine synthetase (IndC) is a single-module NRPS and is involved in the biosynthesis of a bioactive, bright blue dye called Indigoidine from two molecules of L-glutamine in an ATP-dependent reaction. Structural characterization is needed to understand the NRPSs architecture, function, and the communication of NRPSs domains during the synthetic cycle of NRPS, and to facilitate bioengineering to produce new chemical entities. In the present study we cloned a gene, IndC, from thermophilic Streptomyces thermodiastaticus and heterologously expressed it in E. coli BAP1 cell, and then purified the recombinant proteins using standard purification protocol.听Back to Alexandra

Structural Studies of Non-Canonical Aminoglycoside Binding Modes
Aminoglycosides are a broad-spectrum, bactericidal class of antibiotics. Aminoglycoside acetyltransferases (AACs) inactivate aminoglycosides through acetylation. AACs, capture aminoglycosides in a low-energy conformation nearly identical to the binding mode seen in complexes of aminoglycosides bound to bacterial ribosomes, with the aminoglycoside central ring stabilized in chair conformation. This is termed 鈥渢arget mimicry.鈥� We have solved crystal structures of AAC(3)-XIa and AAC(3)-Ia bound to aminoglycosides with their central ring stabilized in boat conformation. These are the first reported structures of resistance enzymes that do not display target mimicry. We hypothesize that these enzymes have a lower affinity to aminoglycosides and that they likely provide less protection against antibiotic treatment. Through isothermal titration calorimetry and in vivo antibiotic susceptibility testing, we assessed the thermodynamic parameters of substrate binding and the practical implications of non-canonical aminoglycoside binding modes. These results provide deeper insights into the role of target mimicry in aminoglycoside resistance.听Back to Mark

Subtle Genomic Differences in Klebsiella pneumoniae May Indicate Adaptation to Distinct Hosts
Capable of causing infections in both dairy cattle and humans, Klebsiella pneumoniae sensu stricto (KpI) may represent a significant zoonotic pathogen. However, the genomic relationship between isolates from bovine and human hosts is unclear. We conducted a comparative genomics analysis using 128 KpI strains from bovine mastitis and human blood infections in Canada. Among these strains, 61 sequence types were identified, and a large accessory genome was found, comprising ~74% of the 15,852 gene unique clusters, indicating high genomic diversity. Human and bovine isolates were intermixed within a core phylogenomic tree, suggesting high genomic similarity, albeit with subtle differences. Bovine strains had significantly more genes associated with ferric citrate iron transporters and utilization of lactose, galactose, galactonate, arginine, and histidine, which may be associated with bovine mastitis. Overall, these findings imply genomic similarity between bovine and human KpI, allowing opportunity for genetic exchange and pathogenic expansion. Back to听Bridget

Synthesis of Inositol Phosphate Analogs Targeting Clostridioides difficile toxin B
Clostridioides difficile infection (CDI) pathogenesis is mainly mediated by secreted protein toxin B (TcdB), whose virulence is modulated by intracellular auto-proteolysis following allosteric activation of its protease domains by myo-inositol hexakisphosphate (IP6). Exogenous administration of IP6 to pre-emptively induce TcdB auto-proteolysis proves ineffective due to its susceptibility to chelate divalent cations found in the gut. Our objective is to design and synthesize new analogs of IP6 and diphospho-myo-inositol pentakisphosphate (IP7) with reduced susceptibility to complexation by divalent cations. We hypothesize our analogs are capable of pre-emptively induce TcdB auto-proteolysis in the colon lumen prior to its uptake into colonic epithelial cells. New analogs of IP6 and IP7 were synthesized by partial replacement of phosphate groups by bisphosphonates, thiophosphates and sulfates. Three thiophosphates and three sulfates on the myo-inositol core improved TcdB cleavage in the presence of divalent cations.听Back to Seyed

TAM complex and outer membrane biogenesis听
Although wastewater treatment plants are propagators of Antimicrobial Resistance Genes (ARGs) in the environment, the ARG dynamics in activated sludge remain unexplained. In this study, reactors received one of two synthetic wastewaters: one simple and readily biodegradable (sugars and volatile fatty acids), and the other complex and slowly biodegradable (proteins and lipids). Upstream sewer influent solids were supplied to simulate immigration.听PCR detected 81 ARGs, and the abundance of several ARGs was significantly higher between the complex and simple feeds (9 and 17 ARGs, respectively) regardless of immigration. Immigration significantly increased the concentrations of 8 genes and decreased the concentration of 4 compared to sludges where no immigration was present, regardless of the feed.听 This study suggests that complex and slowly biodegradable substrates as well as immigration enhance ARG presence and diversity. This means that a greatly polluted wastewater poses a high risk of spreading antimicrobial resistance in the environment.听 Back to Mellouk

Towards a new era in the fight against Campylobacter spp. : Antimicrobial peptides, a promising avenue
The study investigates innovative strategies to combat the rising threat of antibiotic resistance, focusing on five peptides' antimicrobial properties against multidrug-resistant Campylobacter spp., a common cause of foodborne illness. Using whole genome sequencing, researchers aimed to identify resistance and virulence determinants. Results showed significant resistance to ciprofloxacin, tetracycline, ampicillin and gentamicin, with 54.5% of strains expressing multi-resistance., with genomic analysis linking specific genes and mutations to resistance associating tetracycline with the tet(O) gene, ciprofloxacin with mutations in the gyrA gene and in regulatory sequences modulating the expression of an efflux system and aminoglycosides to genes of the aph family. Most strains also carried virulence genes. Peptides MccJ25, MccC, MccB17, and MccE492, produced through fermentation, demonstrated inhibitory effects against the strains, while chemically synthesized Brevi7 showed superior effectiveness. These peptides displayed activity against multidrug-resistant isolates, suggesting their potential as alternatives to traditional antibiotics. The findings underscore Campylobacter's role in spreading antibiotic resistance, emphasizing the urgent need for further research to address this global health concern.听Back to Abdallah

Type 1 fimbriae-mediated collective protection against type 6 secretion system attacks
Bacterial competition may rely on secretion systems such as the type 6 secretion system (T6SS), which punctures and releases toxic molecules into neighboring cells. To subsist, bacterial targets must counteract the threats posed by T6SS-positive competitors. In this study, we used a comprehensive genome-wide high-throughput screening approach to investigate the dynamics of interbacterial competition. Our primary goal was to identify deletion mutants within the well-characterized E. coli K-12 single-gene deletion library, the Keio collection, that demonstrated resistance to T6SS-mediated killing by the enteropathogenic bacterium Cronobacter malonaticus. We identified 49 potential mutants conferring resistance to T6SS and focused our interest on a deletion mutant (鈭唂imE) exhibiting enhanced expression of type 1 fimbriae. We demonstrated that the presence of type 1 fimbriae leads to the formation of microcolonies and thus protects against T6SS-mediated assaults. Collectively, our study demonstrated that adhesive structures such as type 1 fimbriae confer collective protective behavior against T6SS attacks.听Back to Margot

Understanding the role of horizontal gene transfer in the evolution of Acinetobacter
Acinetobacter baumannii can swiftly acquire antimicrobial resistance. Focusing on this species enables us to understand the evolutionary pathways that enable it to be increasingly associated with human infections. We aimed at examining the rate of horizontal gene transfer in a population of clinical Acinetobacter samples.听We constructed the pangenome of A. baumannii clinical genomes. A core gene phylogeny, served as input for ancestral character reconstruction of gene presence/absence.听There is a clear geographic structure in the phylogeny, with the most recently derived lineages corresponding to a single country. Gene gain and loss events are more evident further from the root, suggesting an increasing rate in recent times. The most common 尾-lactamase gene, blaOXA-23, was acquired multiple times independently by different A. baumannii lineages, indicating frequent horizontal gene transfer.听Our results highlight the importance of understanding the evolution of A. baumannii through the lens of lateral gene transfers.听Back to Georgi

Unraveling the crystal structure of the HpaA adhesin: Insights into cell adhesion
To address the spread of antimicrobial resistance (AMR) in the farm-to-fork continuum, we previously developed a real-time surveillance system to monitor the rise of AMR bacteria in the food chain using microfluidic "lab-on-a-chip" for antibiotic susceptibility testing and image recognition. However, this surveillance system only monitors one antimicrobial each time and requires costly GPU that limits its applications in agri-foods. To address this challenge, an embedded AIoT device Orange Pi, priced under $150, was used to control a homemade portable incubator and run an object detection model for identifying chromogenic reactions on multiplex microfluidic chips, which achieved a processing speed exceeding 30 fps and 99% precision in classifying bacterial growth or inhibition. The collected data could be uploaded to the cloud for analysis of large-scale AMR trends. This affordable and compact AI node holds the potential to revolutionize AMR detection across the global food supply chain.听Back to Cyrielle

Utilization of conjugative CRISPR-Cas9 system for targeted elimination of cat and blaCMY-2-bearing plasmid in Escherichia coli from bovine gastrointestinal tract
Escherichia coli is ubiquitous in the dairy setting and disseminated from the intestinal tract to the mammary gland of dairy cows via feces, soil, and bedding resulting in bovine mastitis. Reports of antimicrobial resistant E. coli carrying plasmid-borne antimicrobial resistance genes from bovine mastitis cases raised concerns about antibiotic use and the proliferation of AMR. Here, we used the conjugative plasmid, TP114::Kill1-LC2, that expresses the CRISPR-Cas9 system to eliminate the plasmid, pDJBC01, that harbours chloramphenicol and cefotaxime resistance genes, cat and blaCMY-2. Using a bovine commensal E. coli strain, we created the donor and recipient strains carrying TP114::Kill1-LC2 and pDJBC01, respectively. In vitro conjugation assay showed a substantial pDJBC01 removal rate (87.88-95.82%) with minimal CRISPR-Cas9 escapers (1.7 x 10-5 - 8.8 x 10-6). The in vivo trial of the system in the gastrointestinal tract of Holstein calves will provide its potential to prevent the dissemination of AMR and microbiome modulation.听Back to Dongyun

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MARCC Abstracts by Team

Plant-Based Alternatives for Topical Antibiotics听MARCC Team 2
Tackling antimicrobial resistance (AMR) is a large undertaking that would be impossible to take on all at once. Instead, it should be broken up into smaller, more manageable tasks that, together, can help reduce and eventually solve this problem. The first task that should be addressed, would be to limit the unnecessary use of antimicrobials, as this is one of three main factors that contribute to AMR (1). The other two factors are the widespread use of antimicrobials that are causing a rise in the evolution of resistant phenotypes in microbes, as well as how interconnected the human population is, allowing broad access of pathogens from various places to humanity (1). Topical antibiotics have faced increasing problems of resistant bacteria, as a result of extensive and unchecked use (2). These antibiotics are often used on cuts, burns, and surgical wounds to treat or prevent infections, and while important for more serious issues, for more minor wounds, an alternative approach could be considered (2). Plants have been used for centuries to treat wounds and prevent infections, making them a potential alternative that could limit the overuse of these topical antibiotics (3). There are a variety of plants that possess antimicrobial and antibacterial properties, with a range of activity against different bacteria (3). One plant of note is Calendula officinalis, or pot marigold, whose extracts have been used as an herbal treatment for minor wounds and skin inflammation (3). Extracts of C. officinalis have a number of pharmacological properties, including antibacterial, antimicrobial, antifungal, anti-inflammatory, and antioxidant properties (3). It has also been used to treat bacterial infections in animals (3). Methanol and ethanol extracts of C. officinalis were tested against various pathogens to determine their antimicrobial activity in a previous study, and it was found that both extracts demonstrated good antimicrobial potential (4). [Partial Abstract]听Back to MARCC

Tandem Usage of Genetically Engineered Prophage Therapy and Beta-Lactams in Beta-Lactam Resistant Bacteri MARCC听Team 6
HIV, a provirus that often leads to opportunistic infections in humans³, is a model example of an attack on a biological system on two fronts. The virus replicates and causes damage to the immune system from the inside, consequently raising the susceptibility of the individual to secondary infections. Prophages may be able to set up a similar situation for beta-lactam resistant bacteria. Beta-lactams are one of the largest classes of antibiotics, often used as a last resort due to their broad spectrum effects. However, increases in the prevalence of beta-lactamase producing organisms have been recorded, with resistance levels ranging from 34.28-90.90% of organisms exhibiting resistance depending on the antibiotic used⁶. The primary mechanism of action of these beta-lactam resistant bacteria is the production of inactivation enzymes known as beta-lactamases, which damage the ring structure of beta-lactams, preventing impairment of the cell wall⁵. With disruption to beta-lactamase function through the introduction of new genes coding for beta-lactamase inhibitors via prophages, susceptibility to current antibiotics can be increased, allowing for usage of existing antibiotics rather than relying on the development of new ones. Phages are highly diverse and abundant bacteria-targeting viruses, existing at approximately 10³² particles on Earth⁷. Phage therapy, typically involving bacteriophages in the lytic cycle, is a treatment derived to kill pathogenic bacteria¹. Phages in the lysogenic cycle are not generally used for phage therapy due to prophages frequently encoding bacterial virulence factors⁴. However with CRISPR-Cas9 developments², this investigation hopes to genetically engineer the genome to reduce the genome content to the minimum required for its functionality as a therapeutic vehicle. This investigation aims to increase the efficacy of current beta-lactam antibiotics through the use of a dual phage-antibiotic treatment. Genetic modification of bacterial genomes via prophages can incorporate specific weaknesses into the target organism, providing an opening for antibiotics to work despite the presence of resistance genes. A therapeutic utilizing present industry antibiotics and engineered prophages as a two-front attack on infections can provide aid to the ongoing crisis with minimal effort in development of entirely novel technologies.听Back to MARCC

Guarding the Plate: A Fight against Salmonella Enterica听MARCC听Team 8
Salmonella enterica poses a significant threat to human health, manifesting through a variety of foodborne illnesses. Classified into numerous serotypes, this bacterium can be transmitted through several sources, including animal-derived foods (such as undercooked meat), vegetables, fruits, and other plant products. Its impact on public health has been mainly provoked by the development of antibiotic resistance over the years. Between 1998-2008, Salmonella enterica caused approximately 1.2 million illnesses annually in the United States¹. During this period, it emerged as the leading cause of hospitalization and deaths attributed to foodborne diseases¹. Despite efforts to minimize transmission, another outbreak in 2023 was traced back to cantaloupes in the United States. This incident resulted in 407 reported illnesses, 158 hospitalizations, and 6 deaths². The recurrence of these outbreaks indicates the constant challenges in managing and preventing Salmonella enterica infections². Therefore, it is crucial to discover methods that could weaken the bacterium, aiding the immune system in fighting the infection. A suggested proposition would be to explore possible chemicals for humans to consume that can minimize the bacterium鈥檚 antibiotic resistance. Bacteria such as Salmonella use clever ways to defend themselves from antibiotics, most notably by efflux pumps. Specifically, with the access to the resources, an investigation into the outer membrane protein TolC is what we aim to target for. With TolC being a major component of seven efflux pumps in Salmonella such AcrAB,AcrEf and MdsABC as an outer membrane protein to eject the antibiotics, this would be the ideal component of the efflux pump to target³. We propose two possible outcomes we would like to test regarding this.听[Partial Abstract]听Back to MARCC

Optimizing the botanical innate immune response to bacterial infection using genetically encoded lysins MARCC听Team 9听
Widespread use of antibiotics in agriculture is a predominant contributor to the rise in antimicrobial resistance (AMR) amongst various produce-colonizing bacteria. The Xanthomonas genus is known to cause disease in numerous high-value crops. Xanthomonas vasicola pv. Musacerum (XVM) infection results in Xanthomonas wilt of banana (BXW), which causes the banana plant to rot from the inside out. Here, we propose replacing antibiotics with endolysins, a bacteriophage-originated protein.听Rationale: Unlike antibiotics, endolysins show no signs of contributing to resistance. Previous research has shown that inserting endolysins into the genomes of tomato plants notably reduced bacterial burden, and a broad-scale endolysin targeting gram-negative bacteria (Artilysin) has been engineered. The innate immune system of plants readily responds to damage-associated molecular patterns (DAMPs). Thus, this broad-spectrum endolysin may be encoded in the promoter region of DAMP-related genes.听Methods: The artilysin gene was inserted into the banana plants by Agrobacterium-mediated plant transformation. Cotyledons are selected for successful transformation, and plants are subsequently challenged with XVM. The presence of artilysin is confirmed with polymerase chain reaction (PCR) and immunoblot analysis. Bacterial titer is measured as Colony Forming Units (CFU) per gram of plant material and a survival test is conducted to gauge bacterial viability on transgenic and wild-type plants.听[Partial Abstract]听Back to MARCC

Rethinking How We Prescribe: Using MALDI-TOF MS as a Microbial Diagnostic Tool MARCC听Team 10
As antimicrobial resistance (AMR) is becoming an urgent threat to public health worldwide, clinicians require reliable methods to rapidly identify pathogens in clinical samples and inform initial clinical decision-making regarding antimicrobial prescription. However, current methods of rapid diagnostic testing are costly, can take up to 8 days, and are not commonly used to guide antimicrobial decision-making. A novel diagnostic tool, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), could provide clinicians with same-day microbial strain identification and accurate detection of a pathogen鈥檚 AMR phenotypes. This multi-phase project aims to (i) critically examine the state of MALDI-TOF MS diagnostic testing in a clinical setting to identify challenges and best practices concerning implementation, necessary infrastructures, and clinical outcomes and (ii) assess the influence of MALDI-TOF MS pathogen profiles on clinicians鈥� antimicrobial prescription choices. In collaboration with knowledge users and experts, we will conduct a scoping review of MALDI-TOF MS clinical diagnostic testing by following the methodological steps outlined by Arksey & O鈥橫alley for the conduct of the review. Next, we will conduct an online survey across a representative sample of clinicians from Canadian hospitals and clinics. We will supply clinicians with MALDI-TOF MS data from patients for whom antimicrobials have already been prescribed and document any subsequent prescription changes they would have made. Hopefully, the two phases of this project move us one step closer to achieving our overarching aim of establishing MALDI-TOF MS diagnostic clinical testing preceding antimicrobial prescription as a standard of care throughout Canada鈥檚 healthcare facilities.听Back to MARCC

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