Exploring the depth and breadth of the genomics toolbox during the COVID-19 pandemic: insights from Aotearoa New Zealand.

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.

Antimicrobial Natural Products from Plant Pathogenic Fungi.

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.

The antibacterial potency and antibacterial mechanism of a commercially available surface-anchoring quaternary ammonium salt (SAQAS)-based biocide in vitro.

AIMS: To determine the antimicrobial potency of a surface-anchored quaternary ammonium salt (SAQAS)-based biocide during in vitro wet and dry fomite assays and to determine the mechanism of killing bacteria on the surface. METHODS AND RESULTS: Wet and dry fomite assays were established in vitro for a commercially available biocide (SAQAS-A) applied to glass and low-density polyethylene (LDPE) surfaces. Both wet and dry fomite tests showed the active killing of Gram-positive and Gram-negative bacteria but not endospores. Assays measuring membrane permeability (ATP and DNA release), bacterial membrane potential and bacterial ROS production were correlated with the time-to-kill profiles to show SAQAS-A activity in suspension and applied to a surface. CONCLUSIONS: SAQAS-A is an effective biocide against model strains of vegetative bacteria. The killing mechanism for SAQAS-A observed minimal membrane depolarization, a surge in ROS production and assessment of membrane permeability supported the puncture of cells in both suspension and surface attachment, leading to cell death. SIGNIFICANCE AND IMPACT OF THE STUDY: SAQAS represents effective surface biocides against single challenges with bacteria through a mechanical killing ability that supports real-world application if their durability can be demonstrated to maintain residual activity.

Isolation of a Novel Polyketide from Neodidymelliopsis sp.

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 Polyketide Metabolites from Penicillium bissettii and P. glabrum.

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).

Antimicrobial Metabolites against Methicillin-Resistant Staphylococcus aureus from the Endophytic Fungus Neofusicoccum australe.

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.

A Revised Structure and Assigned Absolute Configuration of Theissenolactone A.

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.

A review on anti-tuberculosis peptides: Impact of peptide structure on anti-tuberculosis activity.

Antibiotic resistance is a major public health problem globally. Particularly concerning amongst drug-resistant human pathogens is Mycobacterium tuberculosis that causes the deadly infectious tuberculosis (TB) disease. Significant issues associated with current treatment options for drug-resistant TB and the high rate of mortality from the disease makes the development of novel treatment options against this pathogen an urgent need. Antimicrobial peptides are part of innate immunity in all forms of life and could provide a potential solution against drug-resistant TB. This review is a critical analysis of antimicrobial peptides that are reported to be active against the M tuberculosis complex exclusively. However, activity on non-TB strains such as Mycobacterium avium and Mycobacterium intracellulare, whenever available, have been included at appropriate sections for these anti-TB peptides. Natural and synthetic antimicrobial peptides of diverse sequences, along with their chemical structures, are presented, discussed, and correlated to their observed antimycobacterial activities. Critical analyses of the structure allied to the anti-mycobacterial activity have allowed us to draw important conclusions and ideas for research and development on these promising molecules to realise their full potential. Even though the review is focussed on peptides, we have briefly summarised the structures and potency of the various small molecule drugs that are available and under development, for TB treatment.

Staphylococcal Protein A Contributes to Persistent Colonization of Mice with Staphylococcus aureus.

Staphylococcus aureus persistently colonizes the nasopharynx in humans, which increases the risk for invasive diseases, such as skin infection and bacteremia. Nasal colonization triggers IgG responses against staphylococcal surface antigens; however, these antibodies cannot prevent subsequent colonization or disease. Here, we describe S. aureus WU1, a multilocus sequence type 88 (ST88) isolate that persistently colonizes the nasopharynx in mice. We report that staphylococcal protein A (SpA) is required for persistence of S. aureus WU1 in the nasopharynx. Compared to animals colonized by wild-type S. aureus, mice colonized with the Δspa variant mount increased IgG responses against staphylococcal colonization determinants. Immunization of mice with a nontoxigenic SpA variant, which cannot cross-link B cell receptors and divert antibody responses, elicits protein A-neutralizing antibodies that promote IgG responses against colonizing S. aureus and diminish pathogen persistence.IMPORTANCEStaphylococcus aureus persistently colonizes the nasopharynx in about one-third of the human population, thereby promoting community- and hospital-acquired infections. Antibiotics are currently used for decolonization of individuals at increased risk of infection. However, the efficacy of antibiotics is limited by recolonization and selection for drug-resistant strains. Here, we propose a model of how staphylococcal protein A (SpA), a B cell superantigen, modifies host immune responses during colonization to support continued persistence of S. aureus in the nasopharynx. We show that this mechanism can be thwarted by vaccine-induced anti-SpA antibodies that promote IgG responses against staphylococcal antigens and diminish colonization.

Global spread of mouse-adapted Staphylococcus aureus lineages CC1, CC15, and CC88 among mouse breeding facilities.

We previously reported that laboratory mice from all global vendors are frequently colonized with Staphylococcus aureus (S. aureus). Genotyping of a snap sample of murine S. aureus isolates from Charles River, US, showed that mice were predominantly colonized with methicillin-sensitive CC88 strains. Here, we expanded our view and investigated whether laboratory mice from other global animal facilities are colonized with similar strains or novel S. aureus lineages, and whether the murine S. aureus isolates show features of host adaptation. In total, we genotyped 230 S. aureus isolates from various vendor facilities of laboratory mice around the globe (Charles River facilities in the USA, Canada, France, and Germany; another US facility) and university- or company-associated breeding facilities in Germany, China and New Zealand. Spa typing was performed to analyse the clonal relationship of the isolates. Moreover, multiplex PCRs were performed for human-specific virulence factors, the immune-evasion cluster (IEC) and superantigen genes (SAg). We found a total of 58 different spa types that clustered into 15 clonal complexes (CCs). Three of these S. aureus lineages had spread globally among laboratory mice and accounted for three quarters of the isolates: CC1 (13.5%), CC15 (14.3%), and CC88 (47.0%). Compared to human colonizing isolates of the same lineages, the murine isolates frequently lacked IEC genes and SAg genes on mobile genetic elements, implying long-term adaptation to the murine host. In conclusion, laboratory mice from various vendors are colonized with host-adapted S. aureus-strains of a few lineages, predominantly the CC88 lineage. S. aureus researchers must be cautioned that S. aureus colonization might be a relevant confounder in infection and vaccination studies and are therefore advised to screen their mice before experimentation.

Do surgical helmet systems affect intraoperative wound contamination? A randomised controlled trial.

BACKGROUND: Deep infection following arthroplasty remains a devastating complication. Some registry data suggests that modern positive-pressure surgical helmet systems (SHS) are associated with a paradoxical increase in infection rates, and as such their role in arthroplasty remains unclear. The aim of this study was to investigate whether SHS increase wound contamination in total knee arthroplasty (TKA) and if this contamination can be reduced by placing tape around the gown/glove interface. METHODS: Seventy-five patients were randomised into three groups: scrubbed theatre staff wore standard surgical gowns (SG), SHS without tape at the gown/glove interface, or SHS with tape. All TKA operations were carried out by the same surgeon. Wound contamination was assessed using a wound culture technique. Blinded laboratory analysis was performed. RESULTS: There were 5/50 culture positive cases when a SHS was used compared to 0/25 when a SG was used; but this difference was not statistically significant (p = 0.16). There were 4/24 culture positive cases when SHS with tape was used compared to 1/26 when SHS without tape was used; but this difference was not statistical significant p = 0.18. CONCLUSION: We found no difference in wound contamination between SG and SHS. Addition of tape at the gown/glove interface did not alter the contamination rate. The choice of surgical gown should take into account cost, comfort and personal protection; as this study found no evidence that wound contamination rates will be altered.

Biologically Active Acetylenic Amino Alcohol and N-Hydroxylated 1,2,3,4-Tetrahydro-ß-carboline Constituents of the New Zealand Ascidian Pseudodistoma opacum.

The first occurrence of an acetylenic 1-amino-2-alcohol, distaminolyne A (1), isolated from the New Zealand ascidian Pseudodistoma opacum, is reported. The isolation and structure elucidation of 1 and assignment of absolute configuration using the exciton coupled circular dichroism technique are described. In addition, a new N-9 hydroxy analogue (2) of the known P. opacum metabolite 7-bromohomotrypargine is also reported. Antimicrobial screening identified modest activity of 1 toward Escherichia coli, Staphylococcus aureus, and Mycobacterim tuberculosis, while 2 exhibited a moderate antimalarial activity (IC50 3.82 µM) toward a chloroquine-resistant strain (FcB1) of Plasmodium falciparum.

Regional Intraosseous Administration of Prophylactic Antibiotics is More Effective Than Systemic Administration in a Mouse Model of TKA.

BACKGROUND: In human TKA studies, intraosseous regional administration (IORA) of prophylactic antibiotics achieves local tissue antibiotic concentrations 10 times greater than systemic administration. However, it is unclear if such high concentrations provide more effective prophylaxis. QUESTIONS/PURPOSES: We asked: (1) What prophylaxis dosage and route (intravenous [IV] versus IORA of prophylactic antibiotics) produce less in vivo bacterial burden compared with no-antibiotic controls? (2) Compared with controls, what prophylaxis dosage and route yield fewer colony-forming units (CFUs) in euthanized animals in a model of TKA? (3) Is prophylactic IORA of antibiotics more effective than same-dose IV antibiotic administration in reducing CFUs? METHODS: Mice (six to nine per group) were block randomized to one of six prophylaxis regimens: control, systemic cefazolin (C100IV), IORA of cefazolin (C100IORA), systemic vancomycin (V110IV), low-dose systemic vancomycin (V25IV), and low-dose IORA of vancomycin (V25IORA). Surgery involved placement of an intraarticular knee prosthesis, followed by an inoculum of bioluminescent Staphylococcus aureus strain Xen36. Biophotonic imaging assessed in vivo bacterial loads, and after 4 days bacterial load was quantified using culture-based techniques. Comparisons were made for each prophylactic regimen to controls and between same-dose IV and IORA of prophylactic antibiotic regimens. RESULTS: Mice treated with systemic high-dose vancomycin, IORA of vancomycin, or IORA of cefazolin had lower in vivo Staphylococcus aureus burdens (median area under curve, Control: 5.0 × 10(6); V110IV: 1.5 × 10(6), difference of medians 3.5 × 10(6), p = 0.003; V25IV: 1.94 × 10(6), difference 3.07 × 10(6), p = 0.49; V25IORA: 1.51 × 10(6), difference 3.5 × 10(6), p = 0.0011; C100IORA: 1.55 × 10(6), difference 3.46 × 10(6), p = 0.0016; C100IV: 2.35 × 10(6), difference 2.66 × 10(6), p = 0.23.) Similar findings were seen with culture-based techniques on recovered implants. IORA of prophylactic antibiotics was more effective than same-dose IV administration in reducing bacterial load on recovered implants (median CFUs < 7.0 × 10(0) vs 2.83 × 10(2), p = 0.0183). CONCLUSIONS: IORA of prophylactic cefazolin and vancomycin was more effective than the same dose of antibiotic given systemically. The effectiveness of vancomycin in particular was enhanced by IORA of prophylactic antibiotics despite using a lower dose. CLINICAL RELEVANCE: Our study supports previous studies of IORA of prophylactic antibiotics in humans and suggests this novel form of administration has the potential to enhance the effectiveness of prophylaxis in TKA. Because of concerns regarding antibiotic stewardship, IORA of prophylactic vancomycin may be more appropriately restricted to patients having TKA who are at greater risk of infection, and clinical trials are in progress.

Clinical and molecular correlates of virulence in Escherichia coli causing bloodstream infection following transrectal ultrasound-guided (TRUS) prostate biopsy.

OBJECTIVES: Prevention and management of Escherichia coli bacteraemia following transrectal ultrasound-guided (TRUS) prostate biopsy has become increasingly complicated by antimicrobial resistance, particularly to fluoroquinolones. Moreover, the globally disseminated, multiresistant sequence type 131 (ST131) E. coli clonal group has recently been described as a major pathogen in the setting of post-biopsy sepsis. Accordingly, we sought to further explore the clinical and molecular epidemiology of post-TRUS biopsy E. coli bacteraemia by comparing the phylogenetic, resistance and virulence characteristics of post-TRUS biopsy E. coli bloodstream isolates with E. coli bloodstream isolates from male patients with spontaneous urosepsis. METHODS: Multiplex PCR was used to compare the phylogenetic group and virulence-associated genes between post-biopsy E. coli isolates and E. coli bloodstream isolates from males with spontaneous urosepsis. Antimicrobial resistance profiles were also compared between the two groups. In addition, we compared the clinical characteristics and outcomes of post-TRUS biopsy patients with E. coli ST131 versus non-ST131 bacteraemia. RESULTS: Although post-TRUS biopsy E. coli isolates were more extensively antimicrobial resistant than isolates from males with spontaneous urosepsis, they harboured significantly fewer virulence-associated genes. In addition, ST131 isolates were significantly less virulent in nature than other isolates from phylogenetic group B2. Clinical outcomes did not differ between patients with post-biopsy ST131 versus non-ST131 bacteraemia. CONCLUSIONS: Our data provide new insights into the molecular pathogenesis of post-TRUS biopsy E. coli bacteraemia, and suggest that antimicrobial resistance, rather than virulence genotype, is the most important bacterial trait associated with an increased risk of infection following TRUS biopsy.

Rapid in vivo assessment of drug efficacy against Mycobacterium tuberculosis using an improved firefly luciferase.

OBJECTIVES: In vivo experimentation is costly and time-consuming, and presents a major bottleneck in anti-tuberculosis drug development. Conventional methods rely on the enumeration of bacterial colonies, and it can take up to 4 weeks for Mycobacterium tuberculosis to grow on agar plates. Light produced by recombinant bacteria expressing luciferase enzymes can be used as a marker of bacterial load, and disease progression can be easily followed non-invasively in live animals by using the appropriate imaging equipment. The objective of this work was to develop a bioluminescence-based mouse model of tuberculosis to assess antibiotic efficacy against M. tuberculosis in vivo. METHODS: We used an M. tuberculosis strain carrying a red-shifted derivative of the firefly luciferase gene (FFlucRT) to infect mice, and monitored disease progression in living animals by bioluminescence imaging before and after treatment with the frontline anti-tuberculosis drug isoniazid. The resulting images were analysed and the bioluminescence was correlated with bacterial counts. RESULTS: Using bioluminescence imaging we detected as few as 1.7 × 10(3) and 7.5 × 10(4) reporter bacteria ex vivo and in vivo, respectively, in the lungs of mice. A good correlation was found between bioluminescence and bacterial load in both cases. Furthermore, a marked reduction in luminescence was observed in living mice given isoniazid treatment. CONCLUSIONS: We have shown that an improved bioluminescent strain of M. tuberculosis can be visualized by non-invasive imaging in live mice during an acute, progressive infection and that this technique can be used to rapidly visualize and quantify the effect of antibiotic treatment. We believe that the model presented here will be of great benefit in early drug discovery as an easy and rapid way to identify active compounds in vivo.

Citrobacter rodentium is an unstable pathogen showing evidence of significant genomic flux.

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.

Population-based incidence and comparative demographics of community-associated and healthcare-associated Escherichia coli bloodstream infection in Auckland, New Zealand, 2005-2011.

BACKGROUND: Escherichia coli is a major human pathogen, both in community and healthcare settings. To date however, relatively few studies have defined the population burden of E. coli bloodstream infections. Such information is important in informing strategies around treatment and prevention of these serious infections. Against this background, we performed a retrospective, population-based observational study of all cases of E. coli bacteremia in patients presenting to our hospital between January 2005 and December 2011. METHODS: Auckland District Health Board is a tertiary-level, university-affiliated institution serving a population of approximately 500,000, within a larger metropolitan population of 1.4 million. We identified all patients with an episode of bloodstream infection due to E. coli over the study period. A unique episode was defined as the first positive E. coli blood culture taken from the same patient within a thirty-day period. Standard definitions were used to classify episodes into community- or healthcare-associated E. coli bacteremia. Demographic information was obtained for all patients, including: age; gender; ethnicity; length of stay (days); requirement for intensive care admission and all-cause, in-patient mortality. RESULTS: A total of 1507 patients had a unique episode of E. coli bacteremia over the study period. The overall average annual incidence of E. coli bacteremia was 52 per 100,000 population, and was highest in the under one year and over 65-year age groups. When stratified by ethnicity, rates were highest in Pacific Peoples and Maori (83 and 62 per 100,000 population respectively). The incidence of community-onset E. coli bacteremia increased significantly over the study period. The overall in-hospital mortality rate was 9% (135/1507), and was significantly higher in patients who had a hospital-onset E. coli bacteremia. CONCLUSIONS: Our work provides valuable baseline data on the incidence of E. coli bacteremia in our locale. The incidence was higher that that described from other developed countries, with significant demographic variation, most notably in ethnic-specific incidence rates. Future work should assess the possible reasons for this disparity.

Inferring bacterial transmission dynamics using deep sequencing genomic surveillance data.

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.

Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence.

OBJECTIVES: Tuberculosis drug development is hampered by the slow growth of Mycobacterium tuberculosis. Bioluminescence, light produced by an enzymatic reaction, constitutes a rapid and highly sensitive measurement of cell metabolic function that can be used as an indirect marker of cell viability in drug screening assays. The aim of this work was to validate and standardize the use of luminescent M. tuberculosis strains to test the activity of antibacterial drugs in vitro and inside macrophages in a 96-well format. METHODS: We have used strains that express the bacterial lux operon and therefore do not require exogenous substrate to produce light, as well as strains expressing the firefly luciferase that need luciferin substrate. Results were compared with those obtained using the resazurin reduction assay and cfu plating. RESULTS: Using bioluminescence we were able to reduce the time required to measure the MIC and bactericidal concentrations of antimicrobials to just 3 and 6 days, respectively. Furthermore, antibacterial activity against intracellular mycobacteria was detected within 2 days post-infection. Results were comparable to those obtained by conventional methods. CONCLUSIONS: We have developed a simple and rapid method for screening antimycobacterial drugs in culture and in macrophages. The use of autoluminescent bacteria also facilitates the determination of growth and inhibition kinetics. The method is cost-effective, can easily be adapted to a larger scale and is amenable to automation. Current efforts are directed towards applying this technology to drug screening in vivo.

A new in vivo model to test anti-tuberculosis drugs using fluorescence imaging.

OBJECTIVES: The current method for testing new drugs against tuberculosis in vivo is the enumeration of bacteria in organs by cfu assay. Owing to the slow growth rate of Mycobacterium tuberculosis (Mtb), these assays can take months to complete. Our aim was to develop a more efficient, fluorescence-based imaging assay to test new antibiotics in a mouse model using Mtb reporter strains. METHODS: A commercial IVIS Kinetic® system and a custom-built laser scanning system with fluorescence molecular tomography (FMT) capability were used to detect fluorescent Mtb in living mice and lungs ex vivo. The resulting images were analysed and the fluorescence was correlated with data from cfu assays. RESULTS: We have shown that fluorescent Mtb can be visualized in the lungs of living mice at a detection limit of ∼8 × 107 cfu/lung, whilst in lungs ex vivo a detection limit of ∼2 × 105 cfu/lung was found. These numbers were comparable between the two imaging systems. Ex vivo lung fluorescence correlated to numbers of bacteria in tissue, and the effect of treatment of mice with the antibiotic moxifloxacin could be visualized and quantified after only 9 days through fluorescence measurements, and was confirmed by cfu assays. CONCLUSIONS: We have developed a new and efficient method for anti-tuberculosis drug testing in vivo, based on fluorescent Mtb reporter strains. Using this method instead of, or together with, cfu assays will reduce the time required to assess the preclinical efficacy of new drugs in animal models and enhance the progress of these candidates into clinical trials against human tuberculosis.