Horizontal gene transfer (HGT) is a key driver of bacterial evolution via transmission of genetic... more Horizontal gene transfer (HGT) is a key driver of bacterial evolution via transmission of genetic materials across taxa. Class 1 integrons are genetic elements that correlate strongly with anthropogenic pollution and contribute to the spread of antimicrobial resistance (AMR) genes via HGT. Despite their significance to human health, there is a shortage of robust, culture-free surveillance technologies for identifying uncultivated environmental taxa that harbour class 1 integrons. We developed a modified version of epicPCR (emulsion,pairedisolation andconcatenationpolymerasechainreaction) that links class 1 integrons amplified from single bacterial cells to taxonomic markers from the same cells in emulsified aqueous droplets. Using this single-cell genomic approach and Nanopore sequencing, we successfully assigned class 1 integron gene cassette arrays containing mostly AMR genes to their hosts in coastal water samples that were affected by pollution. Our work presents the first appli...
Infection triggers a dynamic cascade of reciprocal events between host and pathogen wherein the h... more Infection triggers a dynamic cascade of reciprocal events between host and pathogen wherein the host activates complex mechanisms to recognise and kill pathogens while the pathogen often adjusts its virulence and fitness to avoid eradication by the host. The interaction between the pathogen and the host results in large-scale changes in gene expression in both organisms. Dual RNA-seq, the simultaneous detection of host and pathogen transcripts, has become a leading approach to unravelling complex molecular interactions between the host and the pathogen and is particularly informative for intracellular organisms. The amount of in vitro and in vivo dual RNA-seq data is rapidly growing, which demands computational pipelines to effectively analyse such data. In particular, holistic, systems-level, and temporal analyses of dual RNA-seq data are essential to enable further insights into the host-pathogen transcriptional dynamics and potential interactions. Here, we developed an integrativ...
Acinetobacter baumannii and Klebsiella pneumoniae are opportunistic pathogens frequently co-isola... more Acinetobacter baumannii and Klebsiella pneumoniae are opportunistic pathogens frequently co-isolated from polymicrobial infections. The infections where these pathogens co-exist can be more severe and recalcitrant to therapy than infections caused by either species alone, however there is a lack of knowledge on their potential synergistic interactions. In this study we characterise the genomes of A. baumannii and K. pneumoniae strains co-isolated from a single human lung infection. We examine various aspects of their interactions through transcriptomic, phenomic and phenotypic assays that form a basis for understanding their effects on antimicrobial resistance and virulence during co-infection. Using co-culturing and analyses of secreted metabolites, we discover the ability of K. pneumoniae to cross-feed A. baumannii by-products of sugar fermentation. Minimum inhibitory concentration testing of mono- and co-cultures reveals the ability for A. baumannii to cross-protect K. pneumoniae...
Genotyping studies of Australian Scedosporium isolates have revealed the strong preva-lence of a ... more Genotyping studies of Australian Scedosporium isolates have revealed the strong preva-lence of a recently described species: Scedosporium aurantiacum. In addition to occurring in the environment, this fungus is also known to colonise the respiratory tracts of cystic fibro-sis (CF) patients. A high throughput Phenotype Microarray (PM) analysis using 94 assorted substrates (sugars, amino acids, hexose-acids and carboxylic acids) was carried out for four isolates exhibiting different levels of virulence, determined using aGalleria mellonella infection model. A significant difference was observed in the substrate utilisation patterns of strains displaying differential virulence. For example, certain sugars such as sucrose (sac-charose) were utilised only by low virulence strains whereas some sugar derivatives such as D-turanose promoted respiration only in the more virulent strains. Strains with a higher level of virulence also displayed flexibility and metabolic adaptability at two dif...
Infection triggers a dynamic cascade of reciprocal events between host and pathogen wherein the h... more Infection triggers a dynamic cascade of reciprocal events between host and pathogen wherein the host activates complex mechanisms to recognise and kill pathogens while the pathogen adjusts its virulence and fitness to avoid eradication by the host. The interaction between the pathogen and the host results in large-scale changes in gene expression in both organisms. Dual RNA-seq, the simultaneous detection of host and pathogen transcripts, has become a leading approach to unravel complex molecular interactions between the host and the pathogen and is particularly informative for intracellular organisms. The amount of in vitro and in vivo dual RNA-seq data is rapidly growing which demands computational pipelines to effectively analyse such data. In particular, holistic, systems-level, and temporal analyses of dual RNA-seq data are essential to enable further insights into the host-pathogen transcriptional dynamics and potential interactions. Here, we developed an integrative network-d...
Acinetobacter baumannii and Klebsiella pneumoniae are opportunistic pathogens frequently co-isola... more Acinetobacter baumannii and Klebsiella pneumoniae are opportunistic pathogens frequently co-isolated from polymicrobial infections. The infections where these pathogens co-exist can be more severe and recalcitrant to therapy than infections caused by either species alone, however there is a lack of knowledge on their potential synergistic interactions. In this study we characterised the genomes of A. baumannii and K. pneumoniae strains co-isolated from a single human lung infection. We examined various aspects of their interactions through transcriptomic, phenomic and phenotypic assays that form a basis for understanding their effects on antimicrobial resistance and virulence during co-infection. Using co-culturing and analyses of secreted metabolites, we discovered the ability of K. pneumoniae to cross-feed A. baumannii by-products of sugar fermentation. Minimum inhibitory concentration testing of mono- and co-cultures revealed the ability for A. baumannii to cross-protect K. pneum...
www.PosterPresentations.com S.marcescens, 98.9% Erwinia spp., 0.47% S.marcescens, 95.6% A.xylosox... more www.PosterPresentations.com S.marcescens, 98.9% Erwinia spp., 0.47% S.marcescens, 95.6% A.xylosoxidans, 2.7% Microbiome is the collective genome of microorganisms that inhabit a particular si te. Contact lens (CL) cases become contaminated with microbes during use [1,2] and are often implicated in corneal inf i l t rat ive events [3]. These cult ivable microorganisms represent only a fraction of the microbiota and many bacter ia l s p e c i e s r e m a i n n o n c u l t u r a b l e w i t h standard laboratory techniques. INTRODUCTION
SummaryHorizontal gene transfer between different domains of life is increasingly being recognise... more SummaryHorizontal gene transfer between different domains of life is increasingly being recognised as an important driver of evolution, with the potential to provide the recipient with new gene functionality and assist niche adaptation1-3. However, the molecular mechanisms underlying the integration of exogenous genes from foreign domains are mostly unknown. Integrons are a family of genetic elements that facilitate this process within Bacteria via site-specific DNA recombination4-7. Integrons, however, have not been reported outside Bacteria, and thus their potential role in cross-domain gene transfer has not been investigated. Here we show that integrons are also present among diverse phyla within the domain Archaea. Further, we provide experimental evidence that integron-mediated recombination can facilitate the recruitment of archaeal genes by bacteria. Our findings establish a new mechanism that can facilitate horizontal gene transfer between the two domains of prokaryotes, whi...
Biofilms are organised heterogeneous assemblages of microbial cells that are encased within a sel... more Biofilms are organised heterogeneous assemblages of microbial cells that are encased within a self-produced matrix. Current estimates suggest that up to 80% of bacterial and archaeal cells reside in biofilms. Since biofilms are the main mode of microbial life, understanding their biology and functions is critical, especially as controlling biofilm growth is essential in industrial, infrastructure and medical contexts. Here we discuss biofilms both as collections of individual cells, and as multicellular biological individuals, and introduce the concept of biofilms as unique incubators of diversity for the microbial world.
Integrons were first identified because of their central role in assembling and disseminating ant... more Integrons were first identified because of their central role in assembling and disseminating antibiotic resistance genes in commensal and pathogenic bacteria. However, these clinically relevant integrons represent only a small proportion of integron diversity. Integrons are now known to be ancient genetic elements that are hotspots for genomic diversity, helping to generate adaptive phenotypes. This perspective examines the diversity, functions, and activities of integrons within both natural and clinical environments. We show how the fundamental properties of integrons exquisitely pre-adapted them to respond to the selection pressures imposed by the human use of antimicrobial compounds. We then follow the extraordinary increase in abundance of one class of integrons (class 1) that has resulted from its acquisition by multiple mobile genetic elements, and subsequent colonisation of diverse bacterial species, and a wide range of animal hosts. Consequently, this class of integrons ha...
Integrons are bacterial genetic elements that can integrate mobile gene cassettes. They are mostl... more Integrons are bacterial genetic elements that can integrate mobile gene cassettes. They are mostly known for spreading antibiotic resistance cassettes among human pathogens. However, beyond clinical settings, gene cassettes encode an extraordinarily diverse range of functions important for bacterial adaptation. The recovery and sequencing of cassettes has promising applications, including: surveillance of clinically important genes, particularly antibiotic resistance determinants; investigating the functional diversity of integron-carrying bacteria; and novel enzyme discovery. Although gene cassettes can be directly recovered using PCR, there are no standardised methods for their amplification and, importantly, for validating sequences as genuine integron gene cassettes. Here, we present reproducible methods for the PCR amplification, sequence processing, and validation of gene cassette amplicons from complex communities. We describe two different PCR assays that either amplify cass...
Abstract In this work, we adopt a new tobramycin (TOB)-dopamine coating system to endow thin film... more Abstract In this work, we adopt a new tobramycin (TOB)-dopamine coating system to endow thin film composite membranes with excellent antifouling and antimicrobial properties. Combining the hydrophilic and antibiofouling properties of both TOB and polydopamine, the TOB-dopamine modified membrane exhibits improved antifouling and antimicrobial properties compared with the conventional dopamine modified and unmodified membranes. The TOB-dopamine system has two advantages over the conventional modification with dopamine and tris buffer solution. First, TOB-dopamine modification is more efficient than the conventional dopamine modification due to the accelerating effect of TOB on dopamine polymerization. Second, the TOB-dopamine modified membranes exhibit better hydrophilicity, and enhanced antifouling and antimicrobial properties than the conventional dopamine modified membrane. Beyond engineering membranes, the proposed TOB-dopamine system can also be extended for wider surface hydrophilic and antimicrobial modifications.
Infections caused by Acinetobacter baumannii are increasingly antibiotic resistant, generating a ... more Infections caused by Acinetobacter baumannii are increasingly antibiotic resistant, generating a significant public health problem. Like many bacteria, A. baumannii adopts a biofilm lifestyle that enhances its antibiotic resistance and environmental resilience. Biofilms represent the predominant mode of microbial life, but research into antibiotic resistance has mainly focused on planktonic cells. We investigated the dynamics of A. baumannii biofilms in the presence of antibiotics. A 3-day exposure of A. baumannii biofilms to sub-inhibitory concentrations of antibiotics had a profound effect, increasing biofilm formation and antibiotic resistance in the majority of biofilm dispersal isolates. Cells dispersing from biofilms were genome sequenced to identify mutations accumulating in their genomes, and network analysis linked these mutations to their phenotypes. Transcriptomics of biofilms confirmed the network analysis results, revealing novel gene functions of relevance to both resi...
Pseudomonas protegens Pf-5 (formerly Pseudomonas fluorescens) is a biocontrol bacterium that prod... more Pseudomonas protegens Pf-5 (formerly Pseudomonas fluorescens) is a biocontrol bacterium that produces the siderophore enantio-pyochelin under conditions of iron starvation in a process that is often accompanied by the secretion of its biosynthesis intermediates, salicylic acid and dihydroaeruginoic acid. In this study, we investigated whether several transporters that are encoded by genes within or adjacent to the enantio-pyochelin biosynthetic cluster, serve as efflux systems for enantio-pyochelin and/or its intermediates. In addition, we determined whether these transporters have broad substrates range specificity using a Phenotype Microarray system. Intriguingly, knockouts of the pchH and fetF transporter genes resulted in mutant strains that secrete higher levels of enantio-pyochelin as well as its intermediates salicylic acid and dihydroaeruginoic acid. Analyses of these mutants did not indicate significant change in transcription of biosynthetic genes involved in enantio-pyoch...
Horizontal gene transfer (HGT) is a key driver of bacterial evolution via transmission of genetic... more Horizontal gene transfer (HGT) is a key driver of bacterial evolution via transmission of genetic materials across taxa. Class 1 integrons are genetic elements that correlate strongly with anthropogenic pollution and contribute to the spread of antimicrobial resistance (AMR) genes via HGT. Despite their significance to human health, there is a shortage of robust, culture-free surveillance technologies for identifying uncultivated environmental taxa that harbour class 1 integrons. We developed a modified version of epicPCR (emulsion,pairedisolation andconcatenationpolymerasechainreaction) that links class 1 integrons amplified from single bacterial cells to taxonomic markers from the same cells in emulsified aqueous droplets. Using this single-cell genomic approach and Nanopore sequencing, we successfully assigned class 1 integron gene cassette arrays containing mostly AMR genes to their hosts in coastal water samples that were affected by pollution. Our work presents the first appli...
Infection triggers a dynamic cascade of reciprocal events between host and pathogen wherein the h... more Infection triggers a dynamic cascade of reciprocal events between host and pathogen wherein the host activates complex mechanisms to recognise and kill pathogens while the pathogen often adjusts its virulence and fitness to avoid eradication by the host. The interaction between the pathogen and the host results in large-scale changes in gene expression in both organisms. Dual RNA-seq, the simultaneous detection of host and pathogen transcripts, has become a leading approach to unravelling complex molecular interactions between the host and the pathogen and is particularly informative for intracellular organisms. The amount of in vitro and in vivo dual RNA-seq data is rapidly growing, which demands computational pipelines to effectively analyse such data. In particular, holistic, systems-level, and temporal analyses of dual RNA-seq data are essential to enable further insights into the host-pathogen transcriptional dynamics and potential interactions. Here, we developed an integrativ...
Acinetobacter baumannii and Klebsiella pneumoniae are opportunistic pathogens frequently co-isola... more Acinetobacter baumannii and Klebsiella pneumoniae are opportunistic pathogens frequently co-isolated from polymicrobial infections. The infections where these pathogens co-exist can be more severe and recalcitrant to therapy than infections caused by either species alone, however there is a lack of knowledge on their potential synergistic interactions. In this study we characterise the genomes of A. baumannii and K. pneumoniae strains co-isolated from a single human lung infection. We examine various aspects of their interactions through transcriptomic, phenomic and phenotypic assays that form a basis for understanding their effects on antimicrobial resistance and virulence during co-infection. Using co-culturing and analyses of secreted metabolites, we discover the ability of K. pneumoniae to cross-feed A. baumannii by-products of sugar fermentation. Minimum inhibitory concentration testing of mono- and co-cultures reveals the ability for A. baumannii to cross-protect K. pneumoniae...
Genotyping studies of Australian Scedosporium isolates have revealed the strong preva-lence of a ... more Genotyping studies of Australian Scedosporium isolates have revealed the strong preva-lence of a recently described species: Scedosporium aurantiacum. In addition to occurring in the environment, this fungus is also known to colonise the respiratory tracts of cystic fibro-sis (CF) patients. A high throughput Phenotype Microarray (PM) analysis using 94 assorted substrates (sugars, amino acids, hexose-acids and carboxylic acids) was carried out for four isolates exhibiting different levels of virulence, determined using aGalleria mellonella infection model. A significant difference was observed in the substrate utilisation patterns of strains displaying differential virulence. For example, certain sugars such as sucrose (sac-charose) were utilised only by low virulence strains whereas some sugar derivatives such as D-turanose promoted respiration only in the more virulent strains. Strains with a higher level of virulence also displayed flexibility and metabolic adaptability at two dif...
Infection triggers a dynamic cascade of reciprocal events between host and pathogen wherein the h... more Infection triggers a dynamic cascade of reciprocal events between host and pathogen wherein the host activates complex mechanisms to recognise and kill pathogens while the pathogen adjusts its virulence and fitness to avoid eradication by the host. The interaction between the pathogen and the host results in large-scale changes in gene expression in both organisms. Dual RNA-seq, the simultaneous detection of host and pathogen transcripts, has become a leading approach to unravel complex molecular interactions between the host and the pathogen and is particularly informative for intracellular organisms. The amount of in vitro and in vivo dual RNA-seq data is rapidly growing which demands computational pipelines to effectively analyse such data. In particular, holistic, systems-level, and temporal analyses of dual RNA-seq data are essential to enable further insights into the host-pathogen transcriptional dynamics and potential interactions. Here, we developed an integrative network-d...
Acinetobacter baumannii and Klebsiella pneumoniae are opportunistic pathogens frequently co-isola... more Acinetobacter baumannii and Klebsiella pneumoniae are opportunistic pathogens frequently co-isolated from polymicrobial infections. The infections where these pathogens co-exist can be more severe and recalcitrant to therapy than infections caused by either species alone, however there is a lack of knowledge on their potential synergistic interactions. In this study we characterised the genomes of A. baumannii and K. pneumoniae strains co-isolated from a single human lung infection. We examined various aspects of their interactions through transcriptomic, phenomic and phenotypic assays that form a basis for understanding their effects on antimicrobial resistance and virulence during co-infection. Using co-culturing and analyses of secreted metabolites, we discovered the ability of K. pneumoniae to cross-feed A. baumannii by-products of sugar fermentation. Minimum inhibitory concentration testing of mono- and co-cultures revealed the ability for A. baumannii to cross-protect K. pneum...
www.PosterPresentations.com S.marcescens, 98.9% Erwinia spp., 0.47% S.marcescens, 95.6% A.xylosox... more www.PosterPresentations.com S.marcescens, 98.9% Erwinia spp., 0.47% S.marcescens, 95.6% A.xylosoxidans, 2.7% Microbiome is the collective genome of microorganisms that inhabit a particular si te. Contact lens (CL) cases become contaminated with microbes during use [1,2] and are often implicated in corneal inf i l t rat ive events [3]. These cult ivable microorganisms represent only a fraction of the microbiota and many bacter ia l s p e c i e s r e m a i n n o n c u l t u r a b l e w i t h standard laboratory techniques. INTRODUCTION
SummaryHorizontal gene transfer between different domains of life is increasingly being recognise... more SummaryHorizontal gene transfer between different domains of life is increasingly being recognised as an important driver of evolution, with the potential to provide the recipient with new gene functionality and assist niche adaptation1-3. However, the molecular mechanisms underlying the integration of exogenous genes from foreign domains are mostly unknown. Integrons are a family of genetic elements that facilitate this process within Bacteria via site-specific DNA recombination4-7. Integrons, however, have not been reported outside Bacteria, and thus their potential role in cross-domain gene transfer has not been investigated. Here we show that integrons are also present among diverse phyla within the domain Archaea. Further, we provide experimental evidence that integron-mediated recombination can facilitate the recruitment of archaeal genes by bacteria. Our findings establish a new mechanism that can facilitate horizontal gene transfer between the two domains of prokaryotes, whi...
Biofilms are organised heterogeneous assemblages of microbial cells that are encased within a sel... more Biofilms are organised heterogeneous assemblages of microbial cells that are encased within a self-produced matrix. Current estimates suggest that up to 80% of bacterial and archaeal cells reside in biofilms. Since biofilms are the main mode of microbial life, understanding their biology and functions is critical, especially as controlling biofilm growth is essential in industrial, infrastructure and medical contexts. Here we discuss biofilms both as collections of individual cells, and as multicellular biological individuals, and introduce the concept of biofilms as unique incubators of diversity for the microbial world.
Integrons were first identified because of their central role in assembling and disseminating ant... more Integrons were first identified because of their central role in assembling and disseminating antibiotic resistance genes in commensal and pathogenic bacteria. However, these clinically relevant integrons represent only a small proportion of integron diversity. Integrons are now known to be ancient genetic elements that are hotspots for genomic diversity, helping to generate adaptive phenotypes. This perspective examines the diversity, functions, and activities of integrons within both natural and clinical environments. We show how the fundamental properties of integrons exquisitely pre-adapted them to respond to the selection pressures imposed by the human use of antimicrobial compounds. We then follow the extraordinary increase in abundance of one class of integrons (class 1) that has resulted from its acquisition by multiple mobile genetic elements, and subsequent colonisation of diverse bacterial species, and a wide range of animal hosts. Consequently, this class of integrons ha...
Integrons are bacterial genetic elements that can integrate mobile gene cassettes. They are mostl... more Integrons are bacterial genetic elements that can integrate mobile gene cassettes. They are mostly known for spreading antibiotic resistance cassettes among human pathogens. However, beyond clinical settings, gene cassettes encode an extraordinarily diverse range of functions important for bacterial adaptation. The recovery and sequencing of cassettes has promising applications, including: surveillance of clinically important genes, particularly antibiotic resistance determinants; investigating the functional diversity of integron-carrying bacteria; and novel enzyme discovery. Although gene cassettes can be directly recovered using PCR, there are no standardised methods for their amplification and, importantly, for validating sequences as genuine integron gene cassettes. Here, we present reproducible methods for the PCR amplification, sequence processing, and validation of gene cassette amplicons from complex communities. We describe two different PCR assays that either amplify cass...
Abstract In this work, we adopt a new tobramycin (TOB)-dopamine coating system to endow thin film... more Abstract In this work, we adopt a new tobramycin (TOB)-dopamine coating system to endow thin film composite membranes with excellent antifouling and antimicrobial properties. Combining the hydrophilic and antibiofouling properties of both TOB and polydopamine, the TOB-dopamine modified membrane exhibits improved antifouling and antimicrobial properties compared with the conventional dopamine modified and unmodified membranes. The TOB-dopamine system has two advantages over the conventional modification with dopamine and tris buffer solution. First, TOB-dopamine modification is more efficient than the conventional dopamine modification due to the accelerating effect of TOB on dopamine polymerization. Second, the TOB-dopamine modified membranes exhibit better hydrophilicity, and enhanced antifouling and antimicrobial properties than the conventional dopamine modified membrane. Beyond engineering membranes, the proposed TOB-dopamine system can also be extended for wider surface hydrophilic and antimicrobial modifications.
Infections caused by Acinetobacter baumannii are increasingly antibiotic resistant, generating a ... more Infections caused by Acinetobacter baumannii are increasingly antibiotic resistant, generating a significant public health problem. Like many bacteria, A. baumannii adopts a biofilm lifestyle that enhances its antibiotic resistance and environmental resilience. Biofilms represent the predominant mode of microbial life, but research into antibiotic resistance has mainly focused on planktonic cells. We investigated the dynamics of A. baumannii biofilms in the presence of antibiotics. A 3-day exposure of A. baumannii biofilms to sub-inhibitory concentrations of antibiotics had a profound effect, increasing biofilm formation and antibiotic resistance in the majority of biofilm dispersal isolates. Cells dispersing from biofilms were genome sequenced to identify mutations accumulating in their genomes, and network analysis linked these mutations to their phenotypes. Transcriptomics of biofilms confirmed the network analysis results, revealing novel gene functions of relevance to both resi...
Pseudomonas protegens Pf-5 (formerly Pseudomonas fluorescens) is a biocontrol bacterium that prod... more Pseudomonas protegens Pf-5 (formerly Pseudomonas fluorescens) is a biocontrol bacterium that produces the siderophore enantio-pyochelin under conditions of iron starvation in a process that is often accompanied by the secretion of its biosynthesis intermediates, salicylic acid and dihydroaeruginoic acid. In this study, we investigated whether several transporters that are encoded by genes within or adjacent to the enantio-pyochelin biosynthetic cluster, serve as efflux systems for enantio-pyochelin and/or its intermediates. In addition, we determined whether these transporters have broad substrates range specificity using a Phenotype Microarray system. Intriguingly, knockouts of the pchH and fetF transporter genes resulted in mutant strains that secrete higher levels of enantio-pyochelin as well as its intermediates salicylic acid and dihydroaeruginoic acid. Analyses of these mutants did not indicate significant change in transcription of biosynthetic genes involved in enantio-pyoch...
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