Congratulations to the 2022 Impact AMR Project Prize Winners

Congratulations to the winners of the 2022 Impact AMR Project Prize for best abstracts presented at the Impact AMR ECR and PhD Research Colloquium.

Chris McCory is a PhD student in the lab of Professor Ana Traven.

Investigating the effects of short chain fatty acids on host-fungal pathogen interactions

C. McCrory 1, J. Verma *1, A. Traven *1

1 Monash University, Melbourne, Australia. * The authors marked with an asterisk equally contributed to the work

The human gastrointestinal (GI) tract hosts a diverse microbiome comprising of bacteria, viruses and fungi within complex bionetworks and dynamic microenvironments. Here microbes coexist and compete for nutrients in order to survive. These microbes are fundamental to host digestion and metabolism of dietary macronutrients, producing a vast array of metabolite bi-products, of which short chain fatty acids (SCFAs) are known to have an impact on host and microbial physiology. The yeast Candida albicans is a commensal microbe of the GI tract but can also cause superficial and severe systemic disease. It’s ability to morphologically transition from a yeast to a pathogenic hyphal form is correlated with its clinical manifestation.

We investigate the signalling pathways linking metabolism, gene expression and hyphal morphogenesis of C. albicans. SCFAs are central to these pathways, as they can influence cell biology in multiple ways, including by regulating gene expression via histone acylations. We show that the SCFA crotonate, which regulates the posttranslational modification lysine crotonylation of histones and other proteins, represses hyphal morphogenesis in macrophages and in media mimicking the phagosomal environments, and it also reduces hyphae-dependent macrophage killing by C. albicans. Crotonate has stronger inhibitory effect on hyphal formation compared to the abundant gut SCFA butyrate. RNAseq analysis showed that crotonate inhibits hyphal genes in C. albicans and promotes fatty acid ß-oxidation, which is needed for growth on crotonate as a carbon source. Our current experiments are aimed at understanding how crotonate regulates hyphal morphogenesis, with initial data suggesting that it acts in concert with hyphal transcriptional repressors. Collectively, these studies should shed light on the impact of SCFAs on C. albicans morphogenesis and how the less pathogenic yeast morphology can be promoted over the invasive hyphal morphology.

Jane Hawkey  undertook her PhD in bacterial genomics, focusing on the mobile elements that are responsible for disseminating antimicrobial resistance. During her PhD she developed the software ISMapper for enabling the detection of insertion sequences from short-read sequencing data. She has been a postdoctoral research fellow since 2017 with a focus on the genomic evolution of hospital pathogens. She is particularly interested in developing new genomic methods to track mobile genetic elements that are shared between pathogens, and assess their impact in a hospital setting.

Diversity and transmission of ESBL plasmids in Klebsiella pneumoniae in the hospital setting

Jane Hawkey1, Kelly L Wyres1, Louise M Judd1, Taylor Harshegyi1, Luke Blakeway1, Ryan R Wick1, Adam W J Jenney2, Kathryn E Holt1,3

1 Department of Infectious Diseases, Central Clinical School, Monash University, Victoria, Australia, 2 Microbiology Unit & Department of Infectious Diseases, The Alfred Hospital, Melbourne, Victoria, Australia' 3 London School of Hygiene and Tropical Medicine, London, United Kingdom

Healthcare-associated infections are a top global health priority. Genomics is a powerful approach for investigating transmission of antimicrobial resistant (AMR) infections, however most genomics studies focus on strain, rather than plasmid, transmission. While plasmid transmission is recognised to occur in the hospital setting, the frequency and impact of plasmid transmission on infection burden, compared to ESBL+ strain transmission, is not well understood. From a collection of 104 third-generation cephalosporin resistant K. pneumoniae isolates collected across one year in a large Australian hospital, we used long-read sequencing to complete 70 extended-spectrum beta-lactamase (ESBL) plasmids. We developed methods to detect plasmid transmission events, and found 25 distinct ESBL plasmids in our collection. The majority of ESBL burden during the study period was due to a single plasmid associated with a clonal expansion of K. pneumoniae ST323, plus plasmid transmissions into four additional K. pneumoniae strains. Three of these plasmid positive strains persisted locally 3–6 years later, and we detected this plasmid two additional strain backgrounds. Whilst ESBL plasmid transmission events were rare in this setting, they had a significant and sustained impact on the burden of ceftriaxone-resistant and multidrug-resistant infections. Methods that allow for surveillance of plasmid transmission are key for aiding prevention of transmission of AMR pathogens.

Lenka Vodstrcil is a Senior Research Fellow and the principal epidemiologist in the Genital Microbiota and Mycoplasma Group at the Melbourne Sexual Health Centre,  Central Clinical School, Monash University. Lenka co-leads a translational research program,  aimed at improving management and treatment of the most common vaginal condition, bacterial vaginosis, and treatment and control of sexually transmitted infections, particularly Mycoplasma genitalium. Lenka's research into Mycoplasma genitalium assesses the effectiveness of new drugs and resistance-guided therapy, a strategy that treats patients according to their mycoplasma genitalium drug-resistance profile. This program aims to maintain a high cure rate, and ensure optimal antimicrobial stewardship

Combination Therapy for Mycoplasma genitalium, and New Insights Into the Utility of parC Mutant Detection to Improve Cure

Lenka A Vodstrcil 1 2 3, Erica L Plummer 1 2, Michelle Doyle 1, Gerald L Murray 4 5 6, Kaveesha Bodiyabadu 4 5 6, Jorgen S Jensen 7, David Whiley 8 9, Emma Sweeney 8, Deborah A Williamson 10 11, Eric P F Chow 1 2 3, Christopher K Fairley 1 2, Catriona S Bradshaw 1 2 3

1Melbourne Sexual Health Centre, Alfred Health, Carlton, Victoria, Australia, 2Central Clinical School, Monash University, Melbourne, Victoria, Australia, 3Centre for Epidemiology and Biostatistics, Melbourne School of Population and Global Health, The University of Melbourne, Melbourne, Victoria, Australia, 4Murdoch Children's Research Institute, Parkville, Victoria, Australia, 5Women's Centre for Infectious Diseases, The Royal Women's Hospital, Parkville, Victoria, Australia, 6Department of Obstetrics and Gynaecology, The University of Melbourne, Parkville, Victoria, Australia; 7Research Unit for Reproductive Microbiology, Statens Serum Institut, Copenhagen, Denmark; 8The University of Queensland Centre for Clinical Research (UQ-CCR), Queensland, Australia; 9Pathology Queensland Central Laboratory, Queensland, Australia; 10Department of Microbiology, Royal Melbourne Hospital, Melbourne, Australia ;11Victorian Infectious Diseases Reference Laboratory, The Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia.

Background: Mycoplasma genitalium (MG) infection is challenging to cure because of rising antimicrobial resistance and limited treatment options.

Method: This was a prospective evaluation of the efficacy and tolerability of resistance-guided combination antimicrobial therapy for MG treatment at Melbourne Sexual Health Centre (August 2019–December 2020). All patients received 7 days of doxycycline before combination therapy based on the macrolide-resistant profile. Macrolide-susceptible infections received combination doxycycline + azithromycin (1 g, day 1; 500 mg, days 2–4) and macrolide-resistant infections combination doxycycline + moxifloxacin (400 mg daily for 7 days). Adherence and adverse effects were recorded at test-of-cure, recommended 14–28 days after antimicrobial completion. Sequencing was performed to determine the prevalence of single nucleotide polymorphisms (SNPs) in the parC gene and their association with moxifloxacin treatment outcomes in macrolide-resistant infections.

Results: Of 100 patients with macrolide-susceptible MG treated with doxycycline + azithromycin, 93 were cured (93.0%; 95% confidence interval [CI], 86.1–97.1). Of 247 patients with macrolide-resistant MG receiving doxycycline + moxifloxacin, 210 were cured (85.0%; 95% CI, 80.0–89.2). parC sequencing was available for 164 (66%) macrolide-resistant infections; 29% had SNPs at parC S83 or D87 (23% S83I). The absence of SNPs at parC S83/D87 was associated with 98.3% cure (95% CI, 93.9–99.8) following doxycycline + moxifloxacin. The presence of the parC S83I-SNP was associated with failure in 62.5% (95% CI, 45.8–77.3). Side effects were common (40%–46%) and predominantly mild and gastrointestinal.

Conclusions:Combination doxycycline + azithromycin achieved high cure for macrolide-susceptible infections. However, in the context of a high prevalence of the parC S83I mutation (23%) in macrolide-resistant infections, doxycycline + moxifloxacin cured only 85%. Infections that were wild-type for S83/D87 experienced high cure following doxycycline + moxifloxacin, supporting the use of a parC-resistance/susceptibility testing strategy in clinical care.

Natalia Rosas is currently a PhD candidate in the Department of Microbiology at the Biomedicine Discovery Institute, under Professor Trevor Lithgow supervision. She received her Bachelor degree in microbiology from the Universidad del Valle, Colombia, and her Master’s degree in Biotechnology from the University of Melbourne. Her experience and research interests include molecular biology, microbiology evolution and antimicrobial resistance.Natalia Rosas Bastidas

Investigating the molecular evolution of carbapenem-resistance in Klebsiella quasipneumoniae

Natalia C. Rosas 1,2, Jonathan J. Wilksch 1,2, Jake Barber 1,3, Yanan Wang 1,5, Andrea Rocker2, Laura Perlaza-Jiménez 1,2, Christopher Stubenrauch 1,2, Jiangning Song1,5, George Taiaroa7, Richard A. Strugnell7, Qiyu Bao6, Tieli Zhoud4, Michael J. McDonald1,3 & Trevor Lithgow1,2.

1. Centre to Impact AMR, Monash University, Clayton 3800, Australia; 2. Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton 3800, Australia; 3. School of Biological Sciences, Monash University, Clayton 3800, Australia; 4. The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China;  5. Infection and Immunity Program, Biomedicine Discovery Institute and Department of Biochemistry & Molecular Biology, Monash University, Clayton 3800, Australia; 6. Wenzhou Medical University, Wenzhou, China; 7. Department of Microbiology and Immunology, The Peter Doherty Institute, The University of Melbourne, Parkville 3052, Australia;

The evolution of multidrug resistance in Klebsiella species in response to selective pressure and the extent to which this may be reversible is not fully understood. Since the spread of carbapenem-resistant Enterobacteriaceae (CRE) is considered a public health threat by the World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC), more studies are needed to understand the evolution of antibiotic resistance mechanisms to determine if it is possible to reverse this trend.

A clinical Klebsiella quasipneumoniae isolate called FK688, which caused a bloodstream infection in a hospital patient, is resistant to multiple antibiotics, including third-generation cephalosporins and carbapenems. Analysis of the complete FK688 genome, machine learning and enzymatic analysis revealed no carbapenemase-encoding genes and that epistatic changes are necessary for FK688 to reflect a CRE phenotype.

Evolution experiments demonstrated the fitness burden associated with the antimicrobial resistance determinants and the reversion to a carbapenem-susceptible phenotype in an antibiotic-free environment. Fitness assays showed that a low concentration of ceftazidime selects for a β-lactamase gene and can potentiate evolution to carbapenem resistance by a single-step mutation in the porin OmpK36. The experiments demonstrated the importance of epistatic events and how variation in drug exposure can shape the evolutionary pathway to antibiotic resistance.