Molecular epidemiology of AMR pathogens in Brazil and development of novel control strategies

Abstract

This research project will address two key problems in Sao Paulo state - i) the bacterial crop disease Asiatic Citrus Canker (ACC); and ii) human infections due to multiply antibiotic resistant Klebsiella pneumonia. Our strategy, common to both problems will be to investigate the genomic diversity of the bacterial disease-causing species and to assess the potential of phage encoded capsular polysaccharide depolymerases in protecting from, or curing infection. Brazilian sweet orange production is an industry with key economic and social importance. Orange juice exports to Europe alone are worth in the region of US$32bn annually. Xanthomonas citri subsp. citri (X. citri) is the cause of untreatable citrus canker orange plants that can only be prevented by the identification and destruction of infected plants or spraying with copper biocides. Besides effective, copper biocides are toxic and bio-accumulative. Copper resistance has also emerged in X. citri. We are currently sampling and sequencing X. citri from orchards across the state to characterize the epidemiology of ACC in Sao Paulo. We also have a growing collection of lytic phage, specific for this species whose genomes we are also sequencing to understand their diversity and basis for host specificity. Enright has recently identified several putative capsular polysaccharide depolymerase enzyme genes in these phages that are thought to be involved in hydrolysis of X. citri exo-polysaccharide capsule. This capsule is a major virulence factor that prevents desiccation, promotes host-cell adhesion and tissue invasion and we hypothesize that the application of depolymerases to orange and other citrus plants could prove to be an effective, environmentally sustainable preventative treatment. Our current collaboration is funded by a FAPESP/BBSRC award to examine the epidemiology of X. citri in SP and examine the potential of depolymerases in crop protection (FAPESP 2017/50454-9, and FAPESP 2018/21164-5). This research fellowship will accelerate this work as it will fund a six month visit by Prof Enright to Dr Ferreira's laboratory at UNESP, Rio Claro. In this time he will participate in X. citri collection and he will train UNESP students involved with this and other projects in methods for phage isolation and characterization. He will also be involved in expressing depolymerases and their testing in greenhouse experiments. This research fellowship will also be used to establish a research collaboration targeting the major nosocomial pathogen Klebsiella pneumoniae which is a major cause of multiply antibiotic resistant infections in Brazil. Isolates of some globally disseminated lineages (clones) have evolved resistance to carbapenems - the most effective class of antibiotic for Gram-negative infections. In Brazil some of these carbapenem-resistant K. pneumoniae are also resistant to polymixins which are often described as the "antibiotic of last resort". Enright has established a collection of >100 lytic Klebsiella phages that have demonstrable capsular depolymerase activity against common MDR clones of K. pneumoniae. He has identified 33 separate capsular depolymerases and cloning and expression of some of these enzymes will be carried out as part of his work. Collaborations with scientists in SP hospitals will be initiated during his visit to examine the epidemiology of MDR K. pneumoniae infections in the state. Characterization of lineage and capsular serotype will be performed using whole genome sequencing and MLST and KAPTIVE databases. Genetic associations between host and phage genomes will be investigated to understand the evolutionary dynamics involved in the antagonistic evolution of host capsular polysaccharide capsule genes and those encoding phage capsular polysaccharide depolymerases. This work has great potential to help understand more about host/pathogen interactions and will yield new therapeutic candidates for the treatment of K. pneumonia infections. (AU)