CORD-19:e6e0128e5f23630f1be605cb1a7fb4cc8261f323 JSONTXT 8 Projects

Under the microscope: From pathogens to probiotics and back Under the microscope: From pathogens to probiotics and back Abstract The review centers on the human gastrointestinal tract; focusing first on the bacterial stress responses needed to overcome the physiochemical defenses of the host, specifically how these stress survival strategies can be used as targets for alternative infection control strategies. The concluding section focuses on recent developments in molecular diagnostics; centring on the shifting paradigm from culture to molecular based diagnostics. The unifying theme of my research centers on the microbiology of the gut (more specifically, the human gastrointestinal tract). My interests in this niche map to two main thematic areas (outlined in Fig. 1 ). The first, deals with the elucidation of bacterial stress responses (i.e. the molecular mechanisms enabling bacterial gastrointestinal persistence), and the exploitation of these mechanisms in the design of novel biocontrol strategies. The second, molecular diagnostics, centers on the identification and characterization of new and emerging gastrointestinal pathogens. Herein, I review my contributions in this field. My research career began with a focus on bacterial stress responses; determining how bacteria overcome the various stresses encountered in food production/storage and subsequently, following ingestion, within the gastrointestinal tract. The Gram-positive foodborne bacterium Listeria monocytogenes, a physiologically robust and versatile gastrointestinal pathogen, proved to be the ideal model organism in the early years, which focused on four main stress resistance mechanisms: 1. Acid tolerance -The ability to resist the low pH of pickled foods and the acidic environment of the stomach (»pH 2) 2. Osmotolerance -The ability to grow and survive in dried foods and to resist the low a w of the gastrointestinal tract (with an osmolarity equivalent to 0.3 M NaCl -approximately twice that of blood). 3. Bile tolerance -Resistance to the biological detergent bile, produced in the liver and stored inter-digestively in the gall bladder. As well as aiding the emulsification and digestion of fats, bile forms an important component of the innate defenses of the gut. 4. Barotolerance -the ability to overcome pressures in excess of 300 MPa, associated with high pressure processing of certain foods. The first significant challenge encountered by gastrointestinal pathogens, following ingestion, is the low pH of the stomach. Gastric transit is thus an essential first step in establishing infection. In silico analysis of the L. monocytogenes genome identified lmo0292, a gene predicted to encode an HtrA-like serine protease. 1 Mutation analysis revealed a role for htrA in acid tolerance and virulence potential in L. monocytogenes. Furthermore, transcriptional analysis revealed that htrA is not only induced by low pH stress, but is also under the control of the two-component regulatory system LisRK. 2 Originally identified as an acid tolerance locus and listerial virulence factor, LisRK is also linked to b-lactam resistance in L. monocytogenes. 3 Moreover, we identified LisRK and HtrA as important osmotolerance loci. 2 Indeed, mutational analysis revealed that in addition to regulating HtrA, LisRK likely controls the transcription of one or more additional systems necessary for optimal listerial osmotolerance. Furthermore, it appears that LisRK may act at the initial stages of the listerial osmotic stress-response, with LisK possibly functioning as the primary sensor and LisR (and its genetic targets, e.g. htrA) as the primary respondent(s) to elevated osmolarity. LisRK may thus represent a new dimension in the osmosensing and osmoregulatory capabilities of L. monocytogenes, functioning independently of, but in tandem with, osmolyte uptake synthesis (described below). Osmotolerance L. monocytogenes. In the late 1990s, knowledge of the listerial osmotolerance response was limited; confined mainly to physiological investigations. My strategy was to apply genetic approaches to gain fresh insights into the mechanisms of bacterial osmoadaptation. Indeed, my first published paper 4 described the identification and characterization of BetL, a membrane protein which protects Listeria at elevated osmolarities, by transporting the osmoprotective compound betaine. Representing the first genetic analysis of osmotolerance in Listeria, this study also identified a putative s B -dependent promoter binding site upstream of betL, suggesting that betaine uptake in Listeria might be regulated, at least in part, at the level of transcription. Indeed, we later proved the hypothesis, recording a 1.6-fold increase in betL transcript levels following exposure to elevated osmolarity. 5 Later, while on sabbatical at the laboratory of Prof. Janet Wood at the University of Guelph, Ontario, Canada, I employed a nisin-controlled expression (NICE) system to decouple transcriptional control of betL, revealing that the BetL protein is itself activated (post-translationally) in response to changes in osmolarity. Rapid activation of pre-existing BetL, in response to relatively low NaCl concentrations, suggested that the protein is one of the primary respondents to rapid fluxes in osmolarity. 6 Furthermore, in addition to transcriptional and post-translational control, in silico analysis strongly suggests a role for translational control, not only of betL but of listerial osmoregulation in general. 7 However, despite its obvious role in listerial osmotolerance, deleting betL did not significantly impair growth of the pathogen at elevated osmolarity; suggesting the existence of multiple osmolyte uptake systems. In support of this, we described the identification and characterization of OpuC, a carnitine uptake system which contributes to osmotolerance, chill stress and virulence potential of L. monocytogenes. 8, 9 Indeed, Sleator et al., 8 provided the first direct link between osmotolerance and virulence in Listeria, a link which we ascribed to the accumulation of carnitine, a protective compound similar to betaine but present at much higher concentrations in animal tissues. Following the discovery of Gbu, a third osmolyte uptake system, by Gary Smith's group at the University of California, I undertook a systematic approach to knocking out each of the three uptake systems; BetL, OpuC and Gbu, creating a bank of single, double and triple mutants. This bank allowed me to elucidate, for the first time, the exact role of each transporter against its native background; plotting a hierarchy of osmolyte and transporter importance under osmotic 10 chill stress 11 and desiccation conditions 12 We subsequently pushed the analysis further; 13 establishing the relative importance of each transporter in food and during infection. Interestingly, while betaine appeared to confer most protection in food, the hierarchy of transporter importance differs depending on the food type. While in the animal model, OpuC appears to play the dominant role, with the remaining systems contributing little to the infection process. These findings have had a significant impact on the development of effective control strategies for L. monocytogenes in foods and during infection. In addition to osmolyte uptake, we also identified the first osmolyte synthesis system in L. monocytogenes, proBA 14 While inactivation of proBA had no appreciable effect on virulence, growth at elevated osmolarity was significantly reduced in the absence of efficient proline synthesis. Thus, while proline biosynthesis plays little, if any, role in the intracellular life cycle and infectious nature of L. monocytogenes, it is required for survival in osmolyte-depleted environments of elevated osmolarity. Furthermore, we showed that proline synthesis in Listeria is regulated by feedback inhibition of g-glutamyl kinase, the first enzyme of the proline biosynthesis pathway, encoded by the proB gene. Random saturation mutagenesis of the proBA operon generated 3 independent mutations in proB, leading to proline overproduction. 15 However, as observed for proline auxotrophy, proline hyperproduction has no apparent impact on listerial osmotolerance or indeed virulence potential. Later, using the same random saturation mutagenesis strategy to target betL, we identified a single point mutation in a putative promoter region upstream of betL which led to a dramatic increase in transcript levels under osmo-and chill-stress conditions, and a concomitant increase in stress tolerance. 16 Furthermore, the mutation appears to counter a previously unreported "twisted" cell morphology observed for L. monocytogenes grown at elevated osmolarity. By 2001, my work, and that of others, had fully elucidated the listerial osmotolerance response. 17 In less than 4 years, all of the principal osmolyte uptake and synthesis mechanisms had been identified and characterized. This culminated with the publication of the L. monocyogenes genome sequence which we used to perform a complete in silico analysis of osmotolerance in Listeria. 18 Cronobacter sakazakii. My interest in osmotolerance did not end however with the conclusion of the Listeria story in 2001. Motivated by a 2008 joint United Nations-World Health Organization call, I returned to the subject of osmotolerance, almost a decade later, this time targeting the Gram-negative pathogen Cronobacter sakazakii. C. sakazakii is an extremely osmotolerant gastrointestinal pathogen. 19 The most common route of infection is via powdered infant formula (PIF), resulting in an infant mortality rate of »80%. Unravelling C. sakazakii' s osmotolerance mechanisms is thus a key first step in controlling the pathogen. Unlike the situation with L. monocytogenes a decade earlier, the genome sequence of C. sakazakii was available from the outset. An exhaustive homology transfer based screening strategy, using the Escherichia coli K12 genome as the template, identified 53 putative osmotoleance loci. 20 Interestingly, while C. sakazakii contains homologs of all the principal E. coli osmotolerance loci; a key distinguishing feature is that C. sakazakii possesses multiple copies of certain osmotolerance genes; including seven copies of the E. coli proP homolog, each of which potentially encodes a separate osmolyte uptake system. This level of degeneracy is apparently unique to C. sakazakii (offering a plausible explanation for its distinctive osmotolerance profile). Indeed, gene expression analysis of each of the proP homologs demonstrated an increase in expression levels in response to osmotic upshift. 21 Furthermore, heterologous expression against the osmotically sensitive E. coli MKH13 host revealed that 6 of the 7 homologs conferred an osmotolerance phenotype, albeit to varying degrees. Further structural analysis of the C. sakazakii ProP homologs revealed that all but one (ProP1, which offers the greatest osmoprotective effect) are 60-70 amino acids shorter than the E. coli ProP; lacking the extended carboxyl tail. This immediately suggested a role for the C-terminal domain in modulating the protein's osmoprotective function. This hypothesis was proved by spicing the extended C-terminal domain of ProP1 (encoded by ESA_02131) onto the truncated C-terminal end of ProP2 (encoded by ESA_01706); creating a chimeric protein (ProPc) which exhibits increased osmotolerance relative to the wild type. 22 In tandem with the sequence based approach, functional screening of a C. sakazakii genomic bank, heterologously expressed against the E. coli MKH13 background, revealed a novel role for ProP1 (ESA_02131) as a carnitine uptake system. 23 Indeed, carnitine uptake via ProP1 allows growth at salt concentrations far in excess of that afforded by proline; a finding which has significant food safety implications, given that carnitine is often added to infant formula to boost its nutritional value. Furthermore, we showed that carnitine also promoted the growth of L. monocytoenes in infant formula stored at refrigeration temperatures. 9 Work in my lab has thus not only elucidated the C. sakazakii osmotoleance response, it has also helped to inform food safety policy -calling for a halt to the practice of adding carnitine to infant formula. Furthermore, the osmotolerance mechanisms identified now provide ideal targets for the control of the pathogen. One such approach, involves smugglin technology; 17,18 the use of antimicrobial compounds which structurally mimic carnitine, killing the cell rather than protecting it. This represents a viable alternative to antibiotic therapy and may lead to effective control of the pathogen in high risk foods such as PIF. Metagenomics. In addition to focusing on individual gastrointestinal pathogens, my lab also employs a much broader metagenomics approach to identifying novel osmotolerance loci in the human gut microbiome (reviewed in 24, 25 ). The human distal colon is one of the most complex and densely populated microbial ecosystems on Earth, with cell densities of up to 10 12 per gram of faeces. [26] [27] [28] A functional screen of over 20,000 clones from a human gut microbiota library revealed 53 osmotolerant clones. A combined transposon mutagenesis and bioinformatic strategy revealed three genes (encoding GalE, MurB and MazG) from a single clone, exhibiting high levels of identity to a species from the genus Collinsella. 29 Additionally, in silico analysis of two other clones revealed the presence of an additional galE and mazG gene, from Akkermansia muciniphila and Eggerthella sp., respectively. Each of the three genes identified was found to be over-represented in the human gut metagenome and abundant among healthy subjects from the MetaHit data set, 24 suggesting an important role in gut colonisation. Indeed, as expected, cloning and heterologous expression of the genes in E. coli MKH13 resulted in increased salt tolerance of the transformed cells. A phenotype based microarray analysis identified one of the 53 clones as being positive for utilization/transport of carnitine, 30 while another of the loci was shown to be homologous to a brp/ blh-family b-carotene 15,15 0 -monooxygenase. 31 In addition to the obvious osmotolerance phenotype, E. coli cell pellets expressing brpA adopt a red/orange pigmentation, when cultured in the presence of exogenous b-carotene, indicating the incorporation of carotenoids in the cell membrane. Finally, a significant advantage of functional screens over sequence based approaches is that they provide a powerful means to identify and assign function to novel genes, and their encoded proteins, without any prior sequence knowledge. In support of this, we describe the identification and subsequent analysis of an unknown gene (stlA, for "salt tolerance locus A") with no currently known homologs in the databases. 32 The stlA gene is rare when searched against the human metagenome datasets, MetaHit and the Human Microbiome Project, and represents a novel and unique salt tolerance determinant which is apparently exclusive to the human gut environment. In silico analysis of the L. monocytogenes genome revealed a two-gene operon (formerly opuB), preceded by binding sites for PrfA (positive regulatory factor A) and the alternative stress sigma factor, s B . 18 Despite exhibiting significant sequence similarity to the betaine carnitine choline transporter (BCCT) family, further analysis suggested that the protein most likely functions as a bile efflux pump (actively extruding toxic bile out of the cell), a conclusion substantiated by radiolabelled bile exclusion studies. In addition, functionally inactivating BilE resulted in a significantly increased sensitivity to physiological concentrations of human bile, and a reduction in virulence potential when administered orally to a murine model of infection. 33 Renamed BilE (for Bile Exclusion), this protein not only represented a new and important virulence factor in L. monocytogenes, but also stimulated in me a new interest in bile tolerance as an important gastrointestinal associated virulence mechanism. This was followed by the functional analysis of bsh, pva, and btlBthree genes previously annotated as bile-associated loci in the public databases. 34 While deletion mutants revealed a role for all three genes in resisting the acute toxicity of bile, hydrolysis assays indicated that BSH is likely the only listerial bile salt hydrolase (leading to bile detoxification inside the cell). Transcriptional analyses and activity assays further revealed that, as with bilE, bsh is regulated by both PrfA and s B . The fact that both bilE and bsh are controlled by s B , along with betL, gbu and opuC (which also boasts a PrfA box), suggested a common function for all 4 proteins. 35 In support of this hypothesis, a systematic analysis of strains with mutations in BetL, OpuC and Gbu revealed roles for OpuC, and to a lesser extent BetL, in resisting the acute toxicity of bile. Furthermore, real-time gene expression profiling in the presence of bile, using a lux gene reporter system, revealed that both betL and opuC are induced by bile. 36 Following ingestion, the first physical stress encountered by the bacterium is the low pH of the stomach (»pH 2), followed by elevated osmolarity (equivalent to 0.3M NaCl) and activity of the biological detergent bile in the upper small intestine. Significantly, pre-exposure to elevated osmolarity (0.3 M NaCl for 1 h) resulted in a dramatic increase in the ability of L. monocytogenes to deal with lethal concentrations of bile (adapted cells surviving 1,000 times better than na€ ıve cells 37 ). However, a low-pH challenge (as experienced during gastric transit) fails to similarly protect against subsequent osmotic or bile stress. Equally, preexposure to bile fails to protect against acid or salt. Thus, osmotic stress appears to be at the top of the hierarchy of stress responses during gastrointestinal transit. 38, 39 Given that sigB is transcriptionally upregulated at elevated osmolarity, it is likely that the increased osmolarity of the gastrointestinal lumen may be interpreted by L. monocytogenes as an environmental cue, signaling gut entry (as is the case for Salmonella). 40 Furthermore, given that s B is known to modulate expression of prfA (the master regulator of the virulence gene cluster which coordinates the intracellular phase of L. monocytogenes infection) it is possible that osmotically induced stimulation of the s B regulon in the upper small intestine may not only facilitate successful gastrointestinal transit, but also prime the pathogen for the next phase of infection, which, in susceptible individuals, is the systemic invasive disease listeriosis. 41 Barotolerance Prior to ingestion, gastrointestinal pathogens are often subject to a variety of hostile conditions; particularly stresses associated with food processing. One such stress is high-pressure processing (HPP); a non-thermal food processing method that subjects foods (liquid or solid) to pressures between 50 and 1,000 MPa. Indeed, in Considine et al., 42 we outline how HPP can inactivate microorganisms and enzymes and modify macromolecular structures, with little or no impact on the nutritional and sensory quality of foods. As before, our goal was to identify the genetic loci which allow bacteria to survive HPP; thereby permitting a more in-depth assessment of the implications of HPP in the food industry, both in terms of food quality and safety. A L. monocytogenes genome bank was used to screen for barotolerance loci (i.e., resistance to 400 or 450 MPa for 5 minutes). The study, which represents the first use of agar plates as a novel methodology for screening genome libraries using HPP, identified systems involved in quorum sensing (Agr), flagellar machinery (Mot) and cytokinesis (EzrA) as conferring enhanced pressure resistance when expressed against an E. coli background. 43 In a follow-on study, 44 failure to disrupt EzrA (encoded by lmo1594) revealed that the protein is essential for the growth of L. monocytoenes. Thus, while gene deletion proved impossible; over-expression of lmo1594 confirmed a role for the protein in listerial barotolerance, resulting in significantly improved survival rates at 300 MPa. In an interesting link with previous sections, and substantiating a role for osmolytes as a listerial passe-partout, 45 both osmolyte uptake and synthesis systems have been shown to play a role in listerial barotolerance. Indeed, listerial survival, following exposure to 400 MPa for 5 minutes, increased from 0.008 to 0.02% with added carnitine and to 0.05% with added betaine. 46 While proline synthesis is required for optimal survival when betaine and carnitine are absent or limiting. 47 In 2005 I was appointed as a principal investigator (PI) at the Alimentary Pharmabiotic Center (APC), UCC. With a mission statement "linking Irish science with industry and society through excellence in research, education and outreach in gastrointestinal health," the APC is arguably one of the world's leading centers of gut health research. Indeed, Thomson Reuters Science Watch ranks the APC at number 2 in the world for probiotic research. As an APC PI, I developed an interest in probiotics, 48-50 specifically in applying the tools and techniques developed during my research on gastrointestinal pathogen stress responses, to develop improved probiotics 51 specifically with applications in the developing world. 52, 53 Given the obvious commercial and clinical relevance of probiotic cultures; improving their stress tolerance profile and ability to overcome the physiochemical defenses of the host is, in my opinion, an important biological goal. 54, 55 Patho-biotechnology In 2006 Colin Hill and I coined the term 'patho-biotechnology' to describe the exploitation of pathogen derived stress survival strategies to engineer improved probiotic strains. [56] [57] [58] [59] [60] This approach showed promise not only for the design of more technologically robust probiotic cultures, with improved biotechnological applications, but also in the development of novel vaccine and drug delivery platforms. In support of this, Sheehan et al., 61 describes how heterologous expression of the listerial betL gene in Lactobacillus salivarius significantly increased resistance of the probiotic to several ex vivo stresses, including elevated osmo-, cryo-, baro-, and chill tolerance, as well as increased resistance to spray-and freeze-drying (stresses associated with food processing and preservation). Furthermore, betL also improved in vivo stress survival in Bifidobacterium breve resulting in significantly improved tolerance to gastric juice and osmolarity conditions mimicking the gut environment. Indeed, B. breve strains expressing BetL were recovered at considerably higher levels in the faeces, intestines and cecum of inoculated animals and exhibited significantly lower levels of systemic infection compared to the control strain following oral challenge with L. monocytogenes. 57 Similar results were obtained for both B. breve and Lactococcus lactis when betL was replaced with bilE as the transgene of interest. 62 These physiologically enhanced probiotics function as ideal drug and vaccine delivery platforms; 63 targeting difficult to treat pathogens such as Clostridium difficile, 64 Acinetobacter baumannii 65 and Mycobacteria. [66] [67] [68] In addition to the obvious clinical applications, described above, genetic manipulation of probiotics has provided fundamental insights into their natural life cycle within the host. In Cronin et al., 69 for example, we describe the development of a non-invasive luciferase-based reporter system (pLuxMC1) for real-time tracking of Bifidobacterium species in vivo. This construct allowed us, for the first time, to track the colonisation potential and persistence of the probiotic in real time in live animal models. Indeed, a significant outcome of the study was the identification of the cecum as a niche environment for B. breve; a finding which may explain why appendectomies may lead to an increased risk of functional gastrointestinal disorders. 70 Molecular diagnostics is arguably one of the fastest growing areas in gastrointestinal research. Growth of the so called 'omics' technologies has, over the last decade, led to a gradual migration away from the culture based 'one test, one pathogen' paradigm, toward DNA based multiplex approaches to infectious disease diagnosis, which have in turn led to significant improvements in clinical diagnostics and improved patient care. In 2009 our group established a partnership with Serosep (an Irish molecular diagnostics company) and the Clinical Microbiology Department of Cork University Hospital (CUH), focused on the changing culture of medical microbiology in Ireland. 71 A retrospective analysis of faecal samples submitted to CUH in 2009 was performed to identify all Campylobacter species detected using Serosep's EntericBio Ò multiplex PCR system, in a single calendar year. The results confirmed that routine culture fails to detect over a third of Campylobacter positive samples. From a total of 7,194 diarrheal specimens, 349 Campylobacterpositive samples (23.8%) were shown to be Campylobacter ureolyticus. This represents the first report of C. ureolyticus in the faeces of patients presenting with gastroenteritis, suggesting a heretofore unreported role for this organism as an enteric pathogen. [72] [73] [74] Indeed, we showed that C. ureolyticus is second only to C. jejuni as the most common cause of Campylobacter associated gastroenteritis in Southern Ireland. 75 Furthermore, we reported a prominent seasonal distribution for campylobacteriosis (Spring), with C. ureolyticus (March) preceding C. jejuni/C. coli (April/May) and exhibiting a bimodal age and gender profile; being more commonly detected in elderly female patients. Given that cultural isolation of C. ureolyticus was not possible using the established selective methods for the isolation of Campylobacter spp., we were forced to develop a new selective medium, nalidixic acid amphotericin B vancomycin (NAV), capable of isolating C. ureolyticus from faecal samples. 76 This medium made it possible, for the first time, to isolate pure cultures of C. ureolyticus and to determine the potential source of infections in humans. Building on this, Koziel et al., 77 describes the collection and processing of 164 samples from various domestic animals over a period of 6 months. Significantly, Random Amplified Polymorphic DNA (RAPD) analysis identified a feline derived C. ureolyticus isolate as being genetically similar to a strain (CIT 007) isolated from an elderly female patient presenting with gastroenteritis. Together with the findings of Koziel et al.,, 75 which reported the detection of C. ureolyticus in bovine samples, it appeared likely that this emerging pathogen has a zoonotic potential. Whole genome sequence analysis of CIT 007 appeared to confirm this hypothesis; identifying a number of genetic loci necessary to cause gastrointestinal infection in humans. 78 Indeed, whole genome analysis of two further C. ureolyticus isolates, including the type strain, revealing 106 putative virulence associated factors, 52 of which are predicted to be secreted. Furthermore, this analysis also suggested that C. ureolyticus is likely to exist as a taxonomic continuum comprised of several species (genomospecies) that are likely to have varying impacts on human health and disease. 79 Interestingly, a C. ureolyticus screen in exotic animals led to the identification of strain CIT 045(T) in the faeces of captive lion-tailed macaques (Macaca silenus). Originally believed to be C. ureolyticus on the basis of colony morphology; 16S rRNA analysis showed it to be a completely new Campylobacter species; 80 a finding which we confirmed using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF), together with whole genome sequence analysis. 81 We named this new species Campylobacter corcagiensis sp nov. (The Corkonian!), in honor of its home town of Cork. In parallel with the identification and characterization of C. ureolyticus and C. corcagiensis, our lab has continued to work with Serosep; validating the next two iterations of the Enter-icBio Ò platform. The EntericBio Panel II Ò system, developed to detect Shiga toxin-producing Escherichia coli (STEC), differentiating Shiga-like toxins 1 and 2 (SLT-1 and SLT-2) 82 and the EntericBio real-time Gastro Panel I, which allows real-time detection of C. jejuni, coli, and lari, STEC, Shigella spp, and Salmonella spp. directly from faeces, without pre-enrichment. 83 In addition to gastrointestinal specific bacterial diagnostics, we have also targeted gastrointestinal viruses; namely rotavirus, parvovirus and coronaviruses. Collins et al., 84 86 described a nationwide screen, between 2008 and 2009, of canine parvovirus (CPV) and coronavirus (CCoV); the most common cause of acute gastroenteritis in dogs. A total of 250 faecal samples were collected from both symptomatic and asymptomatic dogs, from a diverse array of sources, representing the major canine groups in Ireland. Seven CPV strains and three CCoV strains were identified in symptomatic dogs, with one animal presenting with mixed CPV/CCoV infection. Significantly, from a public health perspective, each of these animal viruses represents a vast reservoir of genetic material for the diversification of their human equivalents. Thus, a key outcome of these studies is the need for constant genotype surveillance and vigilance. This is particularly relevant given that shifting genotype prevalence, the emergence of novel combinations and mixed infections, are all likely to impact negatively on existing vaccination programmes. In extreme cases this can lead to breakthrough infections. The development of ever faster and cheaper next generation sequencing strategies has resulted in a paradigm shift in molecular diagnostics; changing the focus from single target genes to whole genomes. Since 2008, genomic data has outpaced Moore's Law by a factor of 4, with a global annual sequencing capacity estimated to be 13 quadrillion bases; enough data to fill a stack of DVDs 2 miles high! Recognizing the potential of biology's big data sets in developing improved diagnostic techniques, our lab began to focus on phylogenetics, 87,88 genomics, [89] [90] [91] [92] protein structure/function prediction [93] [94] [95] [96] [97] [98] and the emerging field of synthetic biology. [99] [100] [101] [102] [103] [104] [105] [106] [107] Indeed, this changing focus allowed me to establish links with the Department of Computing at Cork Institute of Technology (CIT). 108, 109 This union produced CIT's first commercial spinout company, nSilico, which I co-founded in 2012. Combining all of the above elements, I wrote and coordinated ClouDx-i, a €1.3 million EU FP7 funded project, with CIT, nSilico and the University of Edinburgh as partners. The principal scientific and technological objective of ClouDx-i was to develop computational approaches to support rapid molecular diagnosis of infection, embedding these techniques in an efficient end-to-end diagnostic process. 110, 111 The project has sequenced and analyzed the genomes of 12 bacterial pathogens, identifying biomarkers for the rapid diagnosis of neonatal infections. [112] [113] [114] [115] [116] [117] [118] No potential conflicts of interest were disclosed.