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Abstract Identifying and interrupting transmission chains is important for controlling infectious diseases. One way to identify transmission pairs – two hosts in which infection was transmitted from one to the other – is using the variation of the pathogen within each single host (within-host variation). However, the role of such variation in transmission is understudied due to a lack of experimental and clinical datasets that capture pathogen diversity in both donor and recipient hosts. In this work, we assess the utility of deep-sequenced genomic surveillance (where genomic regions are sequenced hundreds to thousands of times) using a mouse transmission model involving controlled spread of the pathogenic bacterium Citrobacter rodentium from infected to naïve female animals. We observe that within-host single nucleotide variants (iSNVs) are maintained over multiple transmission steps and present a model for inferring the likelihood that a given pair of sequenced samples are linked by transmission. In this work we show that, beyond the presence and absence of within-host variants, differences arising in the relative abundance of iSNVs (allelic frequency) can infer transmission pairs more precisely. Our approach further highlights the critical role bottlenecks play in reserving the within-host diversity during transmission.

Abstract Background Genomic technologies have become routine in the surveillance and monitoring of the coronavirus disease 2019 (COVID-19) pandemic, as evidenced by the millions of SARS-CoV-2 sequences uploaded to international databases. Yet the ways in which these technologies have been applied to manage the pandemic are varied. Main text Aotearoa New Zealand was one of a small number of countries to adopt an elimination strategy for COVID-19, establishing a managed isolation and quarantine system for all international arrivals. To aid our response, we rapidly set up and scaled our use of genomic technologies to help identify community cases of COVID-19, to understand how they had arisen, and to determine the appropriate action to maintain elimination. Once New Zealand pivoted from elimination to suppression in late 2021, our genomic response changed to focusing on identifying new variants arriving at the border, tracking their incidence around the country, and examining any links between specific variants and increased disease severity. Wastewater detection, quantitation and variant detection were also phased into the response. Here, we explore New Zealand’s genomic journey through the pandemic and provide a high-level overview of the lessons learned and potential future capabilities to better prepare for future pandemics. Conclusions Our commentary is aimed at health professionals and decision-makers who might not be familiar with genetic technologies, how they can be used, and why this is an area with great potential to assist in disease detection and tracking now and in the future.

On 9 March 2020, 2 days before the World Health Organization declared COVID-19 a global pandemic, two of the authors (microbiologist and infectious diseases expert Associate Professor Siouxsie Wiles and cartoonist Toby Morris) released their first output together: an animated GIF (Graphics Interchange Format) known as “Flatten the Curve”. The graphic went viral on Twitter with over 10 million impressions in 3 days. Flatten the Curve was the first of more than 70 graphics produced by our collaboration, all designed as accessible visual communication about COVID-19. The graphics, all released under a Creative Commons CC-BY-SA-4.0 license, have been translated into multiple languages, used by communities, politicians, and public health officials around the world, and the collaborators have won multiple awards for their work.

Antimicrobial compounds, including antibiotics, have been a cornerstone of modern medicine being able to both treat infections and prevent infections in at-risk people, including those who are immune-compromised and those undergoing routine surgical procedures. Their intense use, including in people, animals, and plants, has led to an increase in the incidence of resistant bacteria and fungi, resulting in a desperate need for novel antimicrobial compounds with new mechanisms of action. Many antimicrobial compounds in current use originate from microbial sources, such as penicillin from the fungus Penicillium chrysogenum (renamed by some as P. rubens). Through a collaboration with Aotearoa New Zealand Crown Research Institute Manaaki Whenua–Landcare Research we have access to a collection of thousands of fungal cultures known as the International Collection of Microorganisms from Plants (ICMP). The ICMP contains both known and novel species which have not been extensively tested for their antimicrobial activity. Initial screening of ICMP isolates for activity against Escherichia coli and Staphylococcus aureus directed our interest towards ICMP 477, an isolate of the soil-inhabiting fungus, Aspergillus terreus. In our investigation of the secondary metabolites of A. terreus, through extraction, fractionation, and purification, we isolated nine known natural products. We evaluated the biological activity of selected compounds against various bacteria and fungi and discovered that terrein (1) has potent activity against the important human pathogen Cryptococcus neoformans.

Decontamination of surfaces and items plays an important role in reducing the spread of infectious microorganisms in many settings including hospitals and research institutes. Regardless of the location, appropriate decontamination procedures are required for maintaining biosafety and biosecurity. For example, effective decontamination of microbial cultures is essential to ensure proper biocontainment and safety within microbiological laboratories. To this end, many commercial decontamination agents are available which have been tested to a prescribed standard to substantiate their efficacy. However, these standardised tests are unlikely to accurately reflect many conditions encountered in medical and biomedical research. Despite this, laboratory workers and other users of decontamination agents may assume that all decontamination agents will work in all situations. We tested commonly used commercial decontamination agents against a range of bacterial species to determine their efficacy under real-world research laboratory conditions. As each decontamination agent has a different recommended dilution for use, to compare their efficacy we calculated their ‘effective ratio’ which reflects the difference between the manufacturer-recommended dilution and the dilution needed to achieve decontamination under real-world research laboratory conditions. Effective ratios above one indicate that the agent was effective at a dilution more dilute than recommended whereas effective ratios lower than one indicate that the agent required a higher concentration than recommended. Our results show that the quaternary ammonium agents TriGene Advance and Chemgene HLD4L were the most effective out of the agents tested, with biocidal activity measured at up to 64 times the recommended dilution. In contrast, hypochlorite (bleach) and Prevail™ (stabilised hydrogen peroxide) had the lowest effective ratios amongst the tested agents. In conclusion, our data suggests that not all decontamination agents will work at the recommended dilutions under real-world research laboratory conditions. We recommend that the protocols for the use of decontamination agents are verified under the specific conditions required to ensure they are fit for purpose.

ABSTRACT INTRODUCTIONMass masking is emerging as a key non-pharmaceutical intervention for reducing community spread of COVID-19. However, although hand washing, social distancing and bubble living have been widely adopted by the ‘team of 5 million', mass masking has not been socialised to the general population. AIMTo identify factors associated with face masking in New Zealand during COVID-19 Alert Level 4 lockdown to inform strategies to socialise and support mass masking. METHODSA quantitative online survey conducted in New Zealand during April 2020 invited residents aged ≥18 years to complete a questionnaire. Questions about face masking were included in the survey. The sample was drawn from a commissioned research panel survey, with boosted sampling for Māori and Pacific participants. Responses were weighted to reflect the New Zealand population for all analyses. RESULTSA total of 1015 individuals participated. Self-reported beliefs were strongly related to behaviours, with respondents viewing face masking measures as ‘somewhat' or ‘very' effective in preventing them from contracting COVID-19 more likely to report having worn a face mask than respondents who viewed them as ‘not at all' effective. The strongest barriers to face mask use included beliefs that there was a mask shortage and that the needs of others were greater than their own. DISCUSSIONHighlighting the efficacy of and dispelling myths about the relative efficacy of mask types and socialising people to the purpose of mass masking will contribute to community protective actions of mask wearing in the New Zealand response to COVID-19.

Isolates of a variety of fungal plant pathogens (Alternaria radicina ICMP 5619, Cercospora beticola ICMP 15907, Dactylonectria macrodidyma ICMP 16789, D. torresensis ICMP 20542, Ilyonectria europaea ICMP 16794, and I. liriodendra ICMP 16795) were screened for antimicrobial activity against the human pathogenic bacteria Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Mycobacterium abscessus, and M. marinum and were found to have some activity. Investigation of the secondary metabolites of these fungal isolates led to the isolation of ten natural products (1–10) of which one was novel, (E)-4,7-dihydroxyoct-2-enoic acid (1). Structure elucidation of all natural products was achieved by a combination of NMR spectroscopy and mass spectrometry. We also investigated the antimicrobial activity of a number of the isolated natural products. While we did not find (E)-4,7-dihydroxyoct-2-enoic acid (1) to have any activity against the bacteria and fungi in our assays, we did find that cercosporin (7) exhibited potent activity against Methicillin resistant Staphylococcus aureus (MRSA), dehydro-curvularin (6) and radicicol (10) exhibited antimycobacterial activity against M. marinum, and brefeldin A (8) and radicicol (10) exhibited antifungal activity against Candida albicans. Investigation of the cytotoxicity and haemolytic activities of these natural products (6–8 and 10) found that only one of the four active compounds, radicicol (10), was non-cytotoxic and non-haemolytic.

There is a real and urgent need for new antibiotics able to kill Mycobacteria, acid-fast bacilli capable of causing multiple deadly diseases. These include members of the Mycobacterium tuberculosis complex, which causes the lung disease tuberculosis (TB) as well as non-tuberculous Mycobacteria (NTM) a growing cause of lung, skin, soft tissue, and other infections. Here we describe a medium-throughput bioluminescence-based pipeline to screen fungi for activity against Mycobacteria using the NTM species Mycobacterium abscessus and Mycobacterium marinum. We used this pipeline to screen 36 diverse fungal isolates from the International Collection of Microorganisms from Plants (ICMP) grown on a wide variety of nutrient-rich and nutrient-poor media and discovered that almost all the tested isolates produced considerable anti-mycobacterial activity. Our data also provides strong statistical evidence for the impact of growth media on antibacterial activity. Chemical extraction and fractionation of a subset of the ICMP isolates revealed that much of the activity we observed may be due to the production of the known anti-mycobacterial compound linoleic acid. However, we have identified several ICMP isolates that retained their anti-mycobacterial activity in non-linoleic acid containing fractions. These include isolates of Lophodermium culmigenum, Pseudaegerita viridis, and Trametes coccinea, as well as an unknown species of Boeremia and an isolate of an unknown genus and species in the family Phanerochaetaceae. Investigations are ongoing to identify the sources of their anti-mycobacterial activity and to determine whether any may be due to the production of novel bioactive compounds.

Biofilm infections can be chronic, life threatening and challenging to eradicate. Understanding in vivo stimuli affecting the biofilm cycle is one step toward targeted prevention strategies. Iron restriction by the host is a stimulus for biofilm formation for some Staphylococcus aureus isolates; however, in some infection scenarios bacteria are exposed to abundant amounts of hemoglobin (Hb), which S. aureus is able to use as iron source. Thus, we hypothesized a role for Hb in the biofilm infection. Microplate “biofilm” assays showed biofilm-matrix production was increased in the presence of hemoglobin when compared to the provision of iron as an inorganic salt. Microscopic analysis of biofilms showed that the provision of iron as hemoglobin consistently caused thicker and more structured biofilms when compared to the effect of the inorganic iron source. Iron responsive biofilm gene expression analysis showed that Agr Quorum Sensing, a known biofilm dispersal marker, was repressed with hemoglobin but induced with an equivalent amount of inorganic iron in the laboratory strain Newman. The gene expression of two biofilm structuring agents, PSMα and PSMβ, differed in the response to the iron source provided and was not correlated to hemoglobin-structured biofilms. A comparison of the model pathogen S. aureus Newman with local clinical isolates demonstrated that while there was a similar phenotypic biofilm response to hemoglobin, there was substantial variation in the expression of key biofilm dispersal markers, suggesting an underappreciated variation in biofilm regulome among S. aureus isolates and that no general inferences can be made by studying the behavior of single strains.

Screening of several fungi from the New Zealand International Collection of Microorganisms from Plants identified two strains of Penicillium, P. bissettii and P. glabrum, which exhibited antimicrobial activity against Escherichia coli,Klebsiella pneumoniae, and Staphylococcus aureus. Further investigation into the natural products of the fungi, through extraction and fractionation, led to the isolation of five known polyketide metabolites, penicillic acid (1), citromycetin (2), penialdin A (3), penialdin F (4), and myxotrichin B (5). Semi-synthetic derivatization of 1 led to the discovery of a novel dihydro (1a) derivative that provided evidence for the existence of the much-speculated open-chained form of 1. Upon investigation of the antimicrobial activities of the natural products and derivatives, both penicillic acid (1) and penialdin F (4) were found to inhibit the growth of Methicillin-resistant S. aureus. Penialdin F (4) was also found to have some inhibitory activity against Mycobacterium abscessus and M. marinum along with citromycetin (2).

Lung infection by Mycobacterium tuberculosis is characterized by chronic infection of lung-resident macrophages, long considered to be the primary hosts and determinants of the outcome of the early immune response. Although alveolar macrophages are well-known to host intracellular mycobacteria at later stages of disease, little is known about the earliest events of the innate immune response. The phagocytes that take up mycobacteria immediately following infection, and how the early lung phagocyte response is altered by vaccination with M. bovis bacille Calmette-Guérin (BCG) were unknown. Using BCG expressing the bright red fluorescent protein tdTomato and flow cytometry, we modeled early infection in C57BL/6 mice and tracked phagocyte population kinetics and uptake of mycobacteria, to better understand the involvement of specific phagocyte subsets. By 1 day post-infection, dose-dependent accumulation of neutrophils was observed and surprisingly, granulocytes comprised a greater proportion of infected phagocytes than alveolar macrophages. By 7 days post-infection alveolar macrophages had become the dominant BCG-associated phagocytes. Prior mucosal BCG exposure provided immunized mice with greater frequencies and numbers of lung macrophage subsets, and a significantly greater proportion of alveolar macrophages expressed CD11b prior to and following challenge infection. These data provide the first evidence of granulocytes as the dominant infected phagocyte subset early after mycobacterial infection, and highlight enhanced recruitment of lung macrophages as a factor associated with protection in BCG-immunized mice.

Fungi have become an invaluable source of bioactive natural products, with more than 5 million species of fungi spanning the globe. Fractionation of crude extract of Neodidymelliopsis sp., led to the isolation of a novel polyketide, (2Z)-cillifuranone (1) and five previously reported natural products, (2E)-cillifuranone (2), taiwapyrone (3), xylariolide D (4), pachybasin (5), and N-(5-hydroxypentyl)acetamide (6). It was discovered that (2Z)-cillifuranone (1) was particularly sensitive to ambient temperature and light resulting in isomerisation to (2E)-cillifuranone (2). Structure elucidation of all the natural products were conducted by NMR spectroscopic techniques. The antimicrobial activity of 2, 3, and 5 were evaluated against a variety of bacterial and fungal pathogens. A sodium [1-13C] acetate labelling study was conducted on Neodidymelliopsis sp. and confirmed that pachybasin is biosynthesised through the acetate polyketide pathway.

Antimicrobial bioassay-guided fractionation of the endophytic fungi Neofusicoccum australe led to the isolation of a new unsymmetrical naphthoquinone dimer, neofusnaphthoquinone B (1), along with four known natural products (2–5). Structure elucidation was conducted by nuclear magnetic resonance (NMR) spectroscopic methods, and the antimicrobial activity of all the natural products was investigated, revealing 1 to be moderately active towards methicillin-resistant Staphylococcus aureus (MRSA) with a minimum inhibitory concentration (MIC) of 16 µg/mL.

Natural products have been a great source for drug leads, due to a vast majority possessing unique chemical structures. Such an example is the protoilludane class of natural products which contain an annulated 5/6/4-ring system and are almost exclusively produced by fungi. They have been reported to possess a diverse range of bioactivities, including antimicrobial, antifungal and cytotoxic properties. In this review, we discuss the isolation, structure elucidation and any reported bioactivities of this compound class, including establishment of stereochemistry and any total syntheses of these natural products. A total of 180 protoilludane natural products, isolated in the last 70 years, from fungi, plant and marine sources are covered, highlighting their structural diversity and potential in drug discovery.

Antimicrobial bioassay-guided fractionation of Microcera larvarum led to the isolation of a γ-lactone with a furo[3,4-b]pyran-5-one bicyclic ring system (1) and three known compounds, (3S,4R)-4-hydroxymellein (2), (3S,4S)-4-hydroxymellein (3) and 7-hydroxy-3-(1-hydroxyethyl)isobenzofuran-1(3H)-one (4). Structure elucidation was conducted by NMR spectroscopic methods. Absolute configuration of 1 (2R, 3S, 5S, 7S, 8R) was established using the chiral derivatizing agent MPA and was fully supported by calculated specific rotation and ECD spectra. The spectroscopic data observed for 1 were identical to those previously reported for theissenolactone A (7), necessitating a correction of the latter (from C-5/C-8 trans ring fusion to cis). Compounds 1–4 were evaluated for antimicrobial activity against a panel of pathogens.

Whether mice are an appropriate model for S. aureus infection and vaccination studies is a matter of debate, because they are not considered as natural hosts of S. aureus. We previously identified a mouse-adapted S. aureus strain, which caused infections in laboratory mice. This raised the question whether laboratory mice are commonly colonized with S. aureus and whether this might impact on infection experiments. Publicly available health reports from commercial vendors revealed that S. aureus colonization is rather frequent, with rates as high as 21% among specific-pathogen-free mice. In animal facilities, S. aureus was readily transmitted from parents to offspring, which became persistently colonized. Among 99 murine S. aureus isolates from Charles River Laboratories half belonged to the lineage CC88 (54.5%), followed by CC15, CC5, CC188, and CC8. A comparison of human and murine S. aureus isolates revealed features of host adaptation. In detail, murine strains lacked hlb-converting phages and superantigen-encoding mobile genetic elements, and were frequently ampicillin-sensitive. Moreover, murine CC88 isolates coagulated mouse plasma faster than human CC88 isolates. Importantly, S. aureus colonization clearly primed the murine immune system, inducing a systemic IgG response specific for numerous S. aureus proteins, including several vaccine candidates. Phospholipase C emerged as a promising test antigen for monitoring S. aureus colonization in laboratory mice. In conclusion, laboratory mice are natural hosts of S. aureus and therefore, could provide better infection models than previously assumed. Pre-exposure to the bacteria is a possible confounder in S. aureus infection and vaccination studies and should be monitored.

Tuberculosis is considered a leading cause of death worldwide. More than 95% of cases and deaths occur in low- and middle-income countries. In resource-limited countries, hospitals often lack adequate facilities to manage and isolate patients with infectious tuberculosis (TB), relying instead on personal protective equipment, such as facemasks, to reduce nosocomial transmission of the disease. Facemasks impregnated with an antimicrobial agent may be a cost-effective way of adding an extra level of protection against the spread of TB by reducing the risk of disease transmission. Conducting polymers, such as polyaniline (PANI), and their functionalised derivatives are a novel class of antimicrobial agents with potential as non-leaching additives to provide contamination resistant surfaces. We have investigated the antimicrobial action of PANI and a functionalised derivative, poly-3-aminobenzoic acid (P3ABA), against mycobacteria and have determined the optimal treatment time and concentration to achieve significant knockdown of Mycobacterium smegmatis and Mycobacterium tuberculosis on an agar surface. Results indicated that P3ABA is a potential candidate for use as an anti-tuberculoid agent in facemasks to reduce TB transmission.

Much is known regarding the antibiotic susceptibility of planktonic cultures of Mycobacterium tuberculosis, the bacterium responsible for the lung disease tuberculosis (TB). As planktonically-grown M. tuberculosis are unlikely to be entirely representative of the bacterium during infection, we set out to determine how effective a range of anti-mycobacterial treatments were against M. tuberculosis growing as a biofilm, a bacterial phenotype known to be more resistant to antibiotic treatment. Light levels from bioluminescently-labelled M. tuberculosis H37Rv (strain BSG001) were used as a surrogate for bacterial viability, and were monitored before and after one week of treatment. After treatment, biofilms were disrupted, washed and inoculated into fresh broth and plated onto solid media to rescue any surviving bacteria. We found that in this phenotypic state M. tuberculosis was resistant to the majority of the compounds tested. Minimum inhibitory concentrations (MICs) increased by 20-fold to greater than 1,000-fold, underlying the potential of this phenotype to cause significant problems during treatment.

Bioluminescent reporter genes, such as those from fireflies and bacteria, let researchers use light production as a non-invasive and non-destructive surrogate measure of microbial numbers in a wide variety of environments. As bioluminescence needs microbial metabolites, tagging microorganisms with luciferases means only live metabolically active cells are detected. Despite the wide use of bioluminescent reporter genes, very little is known about the impact of continuous (also called constitutive) light expression on tagged bacteria. We have previously made a bioluminescent strain of Citrobacter rodentium, a bacterium which infects laboratory mice in a similar way to how enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) infect humans. In this study, we compared the growth of the bioluminescent C. rodentium strain ICC180 with its non-bioluminescent parent (strain ICC169) in a wide variety of environments. To understand more about the metabolic burden of expressing light, we also compared the growth profiles of the two strains under approximately 2,000 different conditions. We found that constitutive light expression in ICC180 was near-neutral in almost every non-toxic environment tested. However, we also found that the non-bioluminescent parent strain has a competitive advantage over ICC180 during infection of adult mice, although this was not enough for ICC180 to be completely outcompeted. In conclusion, our data suggest that constitutive light expression is not metabolically costly to C. rodentium and supports the view that bioluminescent versions of microbes can be used as a substitute for their non-bioluminescent parents to study bacterial behaviour in a wide variety of environments.

Siouxsie Wiles reviews by cartoonist Grady Klein and statistician Alan Dabney.

More effective antibiotics and a protective vaccine are desperately needed to combat the 'superbug' Staphylococcus aureus. While in vivo pathogenicity studies routinely involve infection of mice with human S. aureus isolates, recent genetic studies have demonstrated that S. aureus lineages are largely host-specific. The use of such animal-adapted S. aureus strains may therefore be a promising approach for developing more clinically relevant animal infection models. We have isolated a mouse-adapted S. aureus strain (JSNZ) which caused a severe outbreak of preputial gland abscesses among male C57BL/6J mice. We aimed to extensively characterize this strain on a genomic level and determine its virulence potential in murine colonization and infection models. JSNZ belongs to the MLST type ST88, rare among human isolates, and lacks an hlb-converting phage encoding human-specific immune evasion factors. Naive mice were found to be more susceptible to nasal and gastrointestinal colonization with JSNZ than with the human-derived Newman strain. Furthermore, naïve mice required antibiotic pre-treatment to become colonized with Newman. In contrast, JSNZ was able to colonize mice in the absence of antibiotic treatment suggesting that this strain can compete with the natural flora for space and nutrients. In a renal abscess model, JSNZ caused more severe disease than Newman with greater weight loss and bacterial burden. In contrast to most other clinical isolates, JSNZ can also be readily genetically modified by phage transduction and electroporation. In conclusion, the mouse-adapted strain JSNZ may represent a valuable tool for studying aspects of mucosal colonization and for screening novel vaccines and therapies directed at preventing colonization.

Tuberculosis infection, disease and mortality are all less common at high than low altitude and ascent to high altitude was historically recommended for treatment. The immunological and mycobacterial mechanisms underlying the association between altitude and tuberculosis are unclear. We studied the effects of altitude on mycobacteria and antimycobacterial immunity.Antimycobacterial immunity was assayed in 15 healthy adults residing at low altitude before and after they ascended to 3400 meters; and in 47 long-term high-altitude residents. Antimycobacterial immunity was assessed as the extent to which participants' whole blood supported or restricted growth of genetically modified luminescent Bacille Calmette-Guérin (BCG) mycobacteria during 96 hours incubation. We developed a simplified whole blood assay that could be used by a technician in a low-technology setting. We used this to compare mycobacterial growth in participants' whole blood versus positive-control culture broth and versus negative-control plasma.Measurements of mycobacterial luminescence predicted the number of mycobacterial colonies cultured six weeks later. At low altitude, mycobacteria grew in blood at similar rates to positive-control culture broth whereas ascent to high altitude was associated with restriction (p ≤ 0.002) of mycobacterial growth to be 4-times less than in culture broth. At low altitude, mycobacteria grew in blood 25-times more than negative-control plasma whereas ascent to high altitude was associated with restriction (p ≤ 0.01) of mycobacterial growth to be only 6-times more than in plasma. There was no evidence of differences in antimycobacterial immunity at high altitude between people who had recently ascended to high altitude versus long-term high-altitude residents.An assay of luminescent mycobacterial growth in whole blood was adapted and found to be feasible in low-resource settings. This demonstrated that ascent to or residence at high altitude was associated with decreased mycobacterial growth in whole blood relative to controls, consistent with altitude-related augmentation of antimycobacterial cellular immunity.

Streptococcus pyogenes infection of the nasopharynx represents a key step in the pathogenic cycle of this organism and a major focus for vaccine development, requiring robust models to facilitate the screening of potentially protective antigens. One antigen that may be an important target for vaccination is the chemokine protease, SpyCEP, which is cell surface-associated and plays a role in pathogenesis. Biophotonic imaging (BPI) can non-invasively characterize the spatial location and abundance of bioluminescent bacteria in vivo. We have developed a bioluminescent derivative of a pharyngeal S. pyogenes strain by transformation of an emm75 clinical isolate with the luxABCDE operon. Evaluation of isogenic recombinant strains in vitro and in vivo confirmed that bioluminescence conferred a growth deficit that manifests as a fitness cost during infection. Notwithstanding this, bioluminescence expression permitted non-invasive longitudinal quantitation of S. pyogenes within the murine nasopharynx albeit with a detection limit corresponding to approximately 10(5) bacterial colony forming units (CFU) in this region. Vaccination of mice with heat killed streptococci, or with SpyCEP led to a specific IgG response in the serum. BPI demonstrated that both vaccine candidates reduced S. pyogenes bioluminescence emission over the course of nasopharyngeal infection. The work suggests the potential for BPI to be used in the non-invasive longitudinal evaluation of potential S. pyogenes vaccines.

Streptococcus pyogenes is a leading cause of pharyngeal infection, with an estimated 616 million cases per year. The human nasopharynx represents the major reservoir for all S. pyogenes infection, including severe invasive disease. To investigate bacterial and host factors that influence S. pyogenes infection, we have devised an improved murine model of nasopharyngeal colonization, with an optimized dosing volume to avoid fulminant infections and a sensitive host strain. In addition we have utilized a refined technique for longitudinal monitoring of bacterial burden that is non-invasive thereby reducing the numbers of animals required. The model was used to demonstrate that the two component regulatory system, CovR/S, is required for optimum infection and transmission from the nasopharynx. There is a fitness cost conferred by covR/S mutation that is specific to the nasopharynx. This may explain why S. pyogenes with altered covR/S have not become prevalent in community infections despite possessing a selective advantage in invasive infection.

Citrobacter rodentium is a natural mouse pathogen that causes attaching and effacing (A/E) lesions. It shares a common virulence strategy with the clinically significant human A/E pathogens enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) and is widely used to model this route of pathogenesis. We previously reported the complete genome sequence of C. rodentium ICC168, where we found that the genome displayed many characteristics of a newly evolved pathogen. In this study, through PFGE, sequencing of isolates showing variation, whole genome transcriptome analysis and examination of the mobile genetic elements, we found that, consistent with our previous hypothesis, the genome of C. rodentium is unstable as a result of repeat-mediated, large-scale genome recombination and because of active transposition of mobile genetic elements such as the prophages. We sequenced an additional C. rodentium strain, EX-33, to reveal that the reference strain ICC168 is representative of the species and that most of the inactivating mutations were common to both isolates and likely to have occurred early on in the evolution of this pathogen. We draw parallels with the evolution of other bacterial pathogens and conclude that C. rodentium is a recently evolved pathogen that may have emerged alongside the development of inbred mice as a model for human disease.

Mycobacterium tuberculosis, the causative agent of tuberculosis, still represents a major public health threat in many countries. Bioluminescence, the production of light by luciferase-catalyzed reactions, is a versatile reporter technology with multiple applications both in vitro and in vivo. In vivo bioluminescence imaging (BLI) represents one of its most outstanding uses by allowing the non-invasive localization of luciferase-expressing cells within a live animal. Despite the extensive use of luminescent reporters in mycobacteria, the resultant luminescent strains have not been fully applied to BLI.One of the main obstacles to the use of bioluminescence for in vivo imaging is the achievement of reporter protein expression levels high enough to obtain a signal that can be detected externally. Therefore, as a first step in the application of this technology to the study of mycobacterial infection in vivo, we have optimised the use of firefly, Gaussia and bacterial luciferases in mycobacteria using a combination of vectors, promoters, and codon-optimised genes. We report for the first time the functional expression of the whole bacterial lux operon in Mycobacterium tuberculosis and M. smegmatis thus allowing the development of auto-luminescent mycobacteria. We demonstrate that the Gaussia luciferase is secreted from bacterial cells and that this secretion does not require a signal sequence. Finally we prove that the signal produced by recombinant mycobacteria expressing either the firefly or bacterial luciferases can be non-invasively detected in the lungs of infected mice by bioluminescence imaging.While much work remains to be done, the finding that both firefly and bacterial luciferases can be detected non-invasively in live mice is an important first step to using these reporters to study the pathogenesis of M. tuberculosis and other mycobacterial species in vivo. Furthermore, the development of auto-luminescent mycobacteria has enormous ramifications for high throughput mycobacterial drug screening assays which are currently carried out either in a destructive manner using LuxAB or the firefly luciferase.

Fluorescent reporter proteins have proven useful for imaging techniques in many organisms. We constructed optimized expression systems for several fluorescent proteins from the far-red region of the spectrum and analyzed their utility in several mycobacterial species. Plasmids expressing variants of the Discosoma Red fluorescent protein (DsRed) from the Mycobacterium bovis hsp60 promoter were unstable; in contrast expression from the Mycobacterium smegmatis rpsA promoter was stable. In Mycobacterium tuberculosis expression of several of the far-red reporters was readily visualised by eye and three reporters (mCherry, tdTomato, and Turbo-635) fluoresced at a high intensity. Strains expressing mCherry showed no fitness defects in vitro or in macrophages. Treatment of cells with antibiotics demonstrated that mCherry could also be used as a reporter for cell death, since fluorescence decreased in the presence of a bactericidal compound, but remained stable in the presence of a bacteriostatic compound. mCherry was functional under hypoxic conditions; using mCherry we demonstrated that the P(mtbB) is expressed early in hypoxia and progressively down-regulated. mCherry and other far-red fluorescent proteins will have multiple uses in investigating the biology of mycobacteria, particularly under non-replicating, or low cell density conditions, as well as providing a novel means of detecting cell death rapidly.