N-acylhomoserine lactone regulation of adhesion and biofilm differentiation in Serratia marcescens MG1

Serratia marcescens is an opportunistic pathogen involved in predominantly nosocomial infections, however, it is also implicated as a common cause of microbial keratitis. Since many S. marcecens strains are also resistant to multiple antibiotics, this organism represents a growing public health problem. S. marcescens MG1 utilises a regulatory system for regulation of swarming motility and exo-enzyme secretion that relies on the production of a diffusible signal identified as N-butanoyl-L-homoserine lactone (C4-HSL). The aim of this study was to determine the role of C4-HSL in surface colonisation (adhesion and biofilm formation). In this thesis, the development of a novel biofilm in S. marcescens MG1 is described. The biofilm comprises of an intricate and complex structure consisting of long filamentous cells, cell aggregates and cell chains. Two C4-HSL controlled genes (bsmA and bsmB) are shown to be crucial for biofilm formation. It is proposed that C4-HSL regulated bsmA and bsmB gene products are engaged in fine tuning aggregation at a specific time point in late biofilm development. Since adhesion is the first stage of colonisation, the role of C4-HSL in adhesion to a hydrophilic abiotic surface (HAS) and a human corneal epithelial (HCE) cell line was assessed. While adhesion to the HAS was found to be C4-HSL controlled, this was not the case for adhesion to the HCE cells. In adhesion to the HAS, mutations in the following C4-HSL regulated genes resulted in reduced adhesion; a sensor kinase gene (rssA), a type I transporter gene (lipB), bsmA and bsmB. These four genes were found to effect the expression of type I fimbriae which is proposed to be the adhesin affecting C4-HSL regulated adhesion. While C4-HSL is not involved in adhesion to the HCE cell line, the genes bsmA and bsmB are important. It is proposed that bsmA and bsmB dependent HCE adhesion is due to the requirement of these genes for type I fimbriae production. Furthermore, C4-HSL was found to regulate capsule polysaccharide and OmpX production and repress cytotoxic activity against HCE cells and erythrocytes. It is proposed that cytotoxicity is mediated by ShlA haemolysin

The Antimicrobial Resistance Crisis: Causes, Consequences, and Management

The Antimicrobial Resistance (AMR) crisis is the increasing global incidence of infectious diseases affecting the human population, which are untreatable with any known antimicrobial agent. This crisis will have a devastating cost on human society as both debilitating and lethal diseases increase in frequency and scope. Three major factors determine this crisis: 1/ The increasing frequency of AMR phenotypes amongst microbes is an evolutionary response to the widespread use of antimicrobials. 2/ The large and globally connected human population allows pathogens in any environment access to all of humanity. 3/ The extensive and often unnecessary use of antimicrobials by humanity provides the strong selective pressure that is driving the evolutionary response in the microbial world. Of these factors, the size of the human population is least amenable to rapid change. In contrast the remaining two factors may be affected, so offering a means of managing the crisis: The rate at which AMR, as well as virulence factors evolve in microbial world may be slowed by reducing the applied selective pressure. This may be accomplished by radically reducing the global use of current and prospective antimicrobials. Current management measures to legislate the use of antimicrobials and to educate the healthcare world in the issues, while useful, have not comprehensively addressed the problem of achieving an overall reduction in the human use of antimicrobials. We propose that in addition to current measures and increased research into new antimicrobials and diagnostics, a comprehensive education programme will be required to change the public paradigm of antimicrobial usage from that of a first line treatment to that of a last resort when all other therapeutic options have failed

Integration of a laterally acquired gene into a cell network important for growth in a strain of Vibrio rotiferianus

<p>Abstract</p> <p>Background</p> <p>Lateral Gene Transfer (LGT) is a major contributor to bacterial evolution and up to 25% of a bacterium's genome may have been acquired by this process over evolutionary periods of time. Successful LGT requires both the physical transfer of DNA and its successful incorporation into the host cell. One system that contributes to this latter step by site-specific recombination is the integron. Integrons are found in many diverse bacterial Genera and is a genetic system ubiquitous in vibrios that captures mobile DNA at a dedicated site. The presence of integron-associated genes, contained within units of mobile DNA called gene cassettes makes up a substantial component of the vibrio genome (1-3%). Little is known about the role of this system since the vast majority of genes in vibrio arrays are highly novel and functions cannot be ascribed. It is generally regarded that strain-specific mobile genes cannot be readily integrated into the cellular machinery since any perturbation of core metabolism is likely to result in a loss of fitness.</p> <p>Results</p> <p>In this study, at least one mobile gene contained within the <it>Vibrio rotiferianus </it>strain DAT722, but lacking close relatives elsewhere, is shown to greatly reduce host fitness when deleted and tested in growth assays. The precise role of the mobile gene product is unknown but impacts on the regulation of outermembrane porins. This demonstrates that strain specific laterally acquired mobile DNA can be integrated rapidly into bacterial networks such that it becomes advantageous for survival and adaptation in changing environments.</p> <p>Conclusions</p> <p>Mobile genes that are highly strain specific are generally believed to act in isolation. This is because perturbation of existing cell machinery by the acquisition of a new gene by LGT is highly likely to lower fitness. In contrast, we show here that at least one mobile gene, apparently unique to a strain, encodes a product that has integrated into central cellular metabolic processes such that it greatly lowers fitness when lost under those conditions likely to be commonly encountered for the free living cell. This has ramifications for our understanding of the role mobile gene encoded products play in the cell from a systems biology perspective.</p

Writing in a ‘popular science’ style: A paradigm shift

The ability to write professionally and communicate effectively to a range of audiences is considered a fundamental attribute for scientists. To address this, the major assignment in a large second-year university microbiology subject is a written popular science article. Students critically review literature before synthesising and integrating it into a creative, informative narrative for a non-specialised audience. Students find adjusting their writing style and reporting scientific research to suit broader audiences challenging. Previously their articles incorporated too much scientific jargon and lacked creative, accessible narratives. Despite receiving writing instruction in first-year, many students paraphrased, summarised and synthesised the literature inappropriately. Building on the first-year instruction, we designed and embedded targeted scientific writing practices into subject content. Our online interactive tutorials introduce and compare popular and academic science writing conventions. Students also actively practise paraphrasing, summarising and synthesising the literature in an aligned face-to-face workshop. Our evaluation shows an overall improvement in students’ ability to paraphrase, synthesise and adapt the scientific literature to suit the audience. Student engagement with the non-compulsory online tutorials is high and they report increased understanding and confidence in their writing. These results highlight the importance of continuing to teach academic writing in second year and beyond

Gene cassette transcription in a large integron-associated array

<p>Abstract</p> <p>Background</p> <p>The integron/gene cassette system is a diverse and effective adaptive resource for prokaryotes. Short cassette arrays, with less than 10 cassettes adjacent to an integron, provide this resource through the expression of cassette-associated genes by an integron-borne promoter. However, the advantage provided by large arrays containing hundreds of cassettes is less obvious. In this work, using the 116-cassette array of <it>Vibrio </it>sp. DAT722 as a model, we investigated the theory that the majority of genes contained within large cassette arrays are widely expressed by intra-array promoters in addition to the integron-borne promoter.</p> <p>Results</p> <p>We demonstrated that the majority of the cassette-associated genes in the subject array were expressed. We further showed that cassette expression was conditional and that the conditionality varied across the array. We finally showed that this expression was mediated by a diversity of cassette-borne promoters within the array capable of responding to environmental stressors.</p> <p>Conclusions</p> <p>Widespread expression within large gene cassette arrays could provide an adaptive advantage to the host in proportion to the size of the array. Our findings explained the existence and maintenance of large cassette arrays within many prokaryotes. Further, we suggested that repeated rearrangement of cassettes containing genes and/or promoters within large arrays could result in the assembly of operon-like groups of co-expressed cassettes within an array. These findings add to our understanding of the adaptive repertoire of the integron/gene cassette system in prokaryotes and consequently, the evolutionary impact of this system.</p

A genomic island in Vibrio cholerae with VPI-1 site-specific recombination characteristics contains CRISPR-Cas and type VI secretion modules.

Cholera is a devastating diarrhoeal disease caused by certain strains of serogroup O1/O139 Vibrio cholerae. Mobile genetic elements such as genomic islands (GIs) have been pivotal in the evolution of O1/O139 V. cholerae. Perhaps the most important GI involved in cholera disease is the V. cholerae pathogenicity island 1 (VPI-1). This GI contains the toxin-coregulated pilus (TCP) gene cluster that is necessary for colonization of the human intestine as well as being the receptor for infection by the cholera-toxin bearing CTX phage. In this study, we report a GI (designated GIVchS12) from a non-O1/O139 strain of V. cholerae that is present in the same chromosomal location as VPI-1, contains an integrase gene with 94% nucleotide and 100% protein identity to the VPI-1 integrase, and attachment (att) sites 100% identical to those found in VPI-1. However, instead of TCP and the other accessory genes present in VPI-1, GIVchS12 contains a CRISPR-Cas element and a type VI secretion system (T6SS). GIs similar to GIVchS12 were identified in other V. cholerae genomes, also containing CRISPR-Cas elements and/or T6SS's. This study highlights the diversity of GIs circulating in natural V. cholerae populations and identifies GIs with VPI-1 recombination characteristics as a propagator of CRISPR-Cas and T6SS modules

Variability in the Composition of Pacific Oyster Microbiomes Across Oyster Families Exhibiting Different Levels of Susceptibility to OsHV-1 μvar Disease

Oyster diseases are a major impediment to the profitability and growth of the oyster aquaculture industry. In recent years, geographically widespread outbreaks of disease caused by ostreid herpesvirus-1 microvariant (OsHV-1 μvar) have led to mass mortalities among Crassostrea gigas, the Pacific Oyster. Attempts to minimize the impact of this disease have been largely focused on breeding programs, and although these have shown some success in producing oyster families with reduced mortality, the mechanism(s) behind this protection is poorly understood. One possible factor is modification of the C. gigas microbiome. To explore how breeding for resistance to OsHV-1 μvar affects the oyster microbiome, we used 16S rRNA amplicon sequencing to characterize the bacterial communities associated with 35 C. gigas families, incorporating oysters with different levels of susceptibility to OsHV-1 μvar disease. The microbiomes of disease-susceptible families were significantly different to the microbiomes of disease-resistant families. OTUs assigned to the Photobacterium, Vibrio, Aliivibrio, Streptococcus, and Roseovarius genera were associated with low disease resistance. In partial support of this finding, qPCR identified a statistically significant increase of Vibrio-specific 16S rRNA gene copies in the low disease resistance families, possibly indicative of a reduced host immune response to these pathogens. In addition to these results, examination of the core microbiome revealed that each family possessed a small core community, with OTUs assigned to the Winogradskyella genus and the Bradyrhizobiaceae family consistent members across most disease-resistant families. This study examines patterns in the microbiome of oyster families exhibiting differing levels of OsHV-1 μvar disease resistance and reveals some key bacterial taxa that may provide a protective or detrimental role in OsHV-1 μvar disease outbreaks

Crystal Structure of an Integron Gene Cassette-Associated Protein from Vibrio cholerae Identifies a Cationic Drug-Binding Module

Background The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes. Methodology/Principal Findings We report the 1.8 A crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators. Conclusions/Significance Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.National Health and Medical Research Council (Australia) (NHMRC grant 488502)National Institutes of Health (U.S.) (Grant GM62414-0 )Ontario. Ministry of Revenue (Challenge Fund

Antimicrobials : a global alliance for optimizing their rational use in intra-abdominal infections (AGORA)

Intra-abdominal infections (IAI) are an important cause of morbidity and are frequently associated with poor prognosis, particularly in high-risk patients. The cornerstones in the management of complicated IAIs are timely effective source control with appropriate antimicrobial therapy. Empiric antimicrobial therapy is important in the management of intra-abdominal infections and must be broad enough to cover all likely organisms because inappropriate initial antimicrobial therapy is associated with poor patient outcomes and the development of bacterial resistance. The overuse of antimicrobials is widely accepted as a major driver of some emerging infections (such as C. difficile), the selection of resistant pathogens in individual patients, and for the continued development of antimicrobial resistance globally. The growing emergence of multi-drug resistant organisms and the limited development of new agents available to counteract them have caused an impending crisis with alarming implications, especially with regards to Gram-negative bacteria. An international task force from 79 different countries has joined this project by sharing a document on the rational use of antimicrobials for patients with IAIs. The project has been termed AGORA (Antimicrobials: A Global Alliance for Optimizing their Rational Use in Intra-Abdominal Infections). The authors hope that AGORA, involving many of the world's leading experts, can actively raise awareness in health workers and can improve prescribing behavior in treating IAIs.Peer reviewe