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In this study, we report the semisynthesis and in vitro biological evaluation of thirty-four derivatives of the fungal depsidone antibiotic, unguinol. Initially, the semisynthetic modifications were focused on the two free hydroxy groups (3-OH and 8-OH), the three free aromatic positions (C-2, C-4 and C-7), the butenyl side chain and the depsidone ester linkage. Fifteen first-generation unguinol analogues were synthesised and screened against a panel of bacteria, fungi and mammalian cells to formulate a basic structure activity relationship (SAR) for the unguinol pharmacophore. Based on the SAR studies, we synthesised a further nineteen second-generation analogues, specifically aimed at improving the antibacterial potency of the pharmacophore. In vitro antibacterial activity testing of these compounds revealed that 3-O-(2-fluorobenzyl)unguinol and 3-O-(2,4-difluorobenzyl)unguinol showed potent activity against both methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MIC 0.25–1 μg mL−1) and are promising candidates for further development in vivo.

One approach to combat the increasing incidence of multidrug-resistant (MDR) bacterial pathogens involves repurposing existing compounds with known safety and development pathways as new antibacterial classes with potentially novel mechanisms of action. Here, triclabendazole (TCBZ), a drug originally developed to treat Fasciola hepatica (liver fluke) in sheep and cattle, and later in humans, was evaluated as an antibacterial alone or in combination with sub-inhibitory concentrations of polymyxin B (PMB) against clinical isolates and reference strains of key Gram-positive and Gram-negative bacteria. We show for the first time that in vitro, TCBZ selectively kills methicillin-sensitive and methicillin-resistant Staphylococcus aureus and Staphylococcus pseudintermedius at a minimum inhibitory concentration (MIC) range of 2–4 µg/mL, and vancomycin-resistant enterococci at a MIC range of 4–8 µg/mL. TCBZ also inhibited key Gram-negative bacteria in the presence of sub-inhibitory concentrations of PMB, returning MIC90 values of 1 µg/mL for Escherichia coli, 8 µg/mL for Klebsiella pneumoniae, 2 µg/mL for Acinetobacter baumannii and 4 µg/mL for Pseudomonasaeruginosa. Interestingly, TCBZ was found to be bacteriostatic against intracellular S. aureus but bactericidal against intracellular S. pseudintermedius. Additionally, TCBZ’s favourable pharmacokinetic (PK) and pharmacodynamic (PD) profile was further explored by in vivo safety and efficacy studies using a bioluminescent mouse model of S. aureus sepsis. We show that repeated four-hourly oral treatment of mice with 50 mg/kg TCBZ after systemic S. aureus challenge resulted in a significant reduction in S. aureus populations in the blood to 18 h post-infection (compared to untreated mice) but did not clear the bacterial infection from the bloodstream, consistent with in vivo bacteriostatic activity. These results indicate that additional pharmaceutical development of TCBZ may enhance its PK/PD, allowing it to be an appropriate candidate for the treatment of serious MDR bacterial pathogens.

Multiplex assays for malaria antigen detection can gather data from large sample sets, but considerations for the consistency and quality assurance (QA) of mass testing lack evaluation. We present a QA framework for a study occurring November 2019 to March 2020 involving 504 assay plates detecting four Plasmodium antigens: pan-Plasmodium aldolase and lactate dehydrogenase (LDH), histidine-rich protein 2 (HRP2), P. vivax LDH (PvLDH). Controls on each plate included buffer blank, antigen negative blood, and 4-point positive dilution curve. The blank and negative blood provided consistently low signal for all targets except for pAldolase, which showed variability. Positive curve signals decreased throughout the 5-month study duration but retained a coefficient of variation (CV) of

Our recent focus on the “lost antibiotic” unguinol and related nidulin-family fungal natural products identified two semisynthetic derivatives, benzguinols A and B, with unexpected in vitro activity against Staphylococcus aureus isolates either susceptible or resistant to methicillin. Here, we show further activity of the benzguinols against methicillin-resistant isolates of the animal pathogen Staphylococcus pseudintermedius, with minimum inhibitory concentration (MIC) ranging 0.5–1 μg/mL. When combined with sub-inhibitory concentrations of colistin, the benzguinols demonstrated synergy against Gram-negative reference strains of Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa (MICs of 1–2 μg/mL in the presence of colistin), whereas the benzguinols alone had no activity. Administration of three intraperitoneal (IP) doses of 20 mg/kg benzguinol A or B to mice did not result in any obvious adverse clinical or pathological evidence of acute toxicity. Importantly, mice that received three 20 mg/kg IP doses of benzguinol A or B at 4 h intervals exhibited significantly reduced bacterial loads and longer survival times than vehicle-only treated mice in a bioluminescent S. aureus murine sepsis challenge model. We conclude that the benzguinols are potential candidates for further development for specific treatment of serious bacterial infections as both stand-alone antibiotics and in combination with existing antibiotic classes.

In this study, we optimized and compared different transmission electron microscopy (TEM) methods to visualize changes to Gram-negative bacterial morphology induced by treatment with a robenidine analogue (NCL195) and colistin combination. Aldehyde-fixed bacterial cells (untreated, treated with colistin or NCL195 + colistin) were prepared using conventional TEM methods and compared with ultrathin Tokuyasu cryo-sections. The results of this study indicate superiority of ultrathin cryo-sections in visualizing the membrane ultrastructure of Escherichia coli and Pseudomonas aeruginosa, with a clear delineation of the outer and inner membrane as well as the peptidoglycan layer. We suggest that the use of ultrathin cryo-sectioning can be used to better visualize and understand drug interaction mechanisms on the bacterial cell membrane. Refereed/Peer-reviewed

Pre-harvest sanitation of irrigation water has potential for reducing pathogen contamination of fresh produce. We compared the sanitizing effects of irrigation water containing neutral electrolyzed oxidizing water (EOW) or sodium hypochlorite (NaClO) on pre-harvest lettuce and baby spinach leaves artificially contaminated with a mixture of Escherichia coli, Salmonella Enteritidis and Listeria innocua (∼1 × 108 colony-forming units/mL each resuspended in water containing 100 mg/L dissolved organic carbon, simulating a splash-back scenario from contaminated soil/manure). The microbial load and leaf quality were assessed over 7 days, and post-harvest shelf life evaluated for 10 days. Irrigation with water containing EOW or NaClO at 50 mg/L free chlorine significantly reduced the inoculated bacterial load by ≥ 1.5 log10, whereas tap water irrigation reduced the inoculated bacterial load by an average of 0.5 log10, when compared with untreated leaves. There were no visual effects of EOW or tap water irrigation on baby spinach or lettuce leaf surfaces pre- or post-harvest, whereas there were obvious negative effects of NaClO irrigation on leaf appearance for both plants, including severe necrotic zones and yellowing/browning of leaves. Therefore, EOW could serve as a viable alternative to chemical-based sanitizers for pre-harvest disinfection of minimally processed vegetables. Refereed/Peer-reviewed

The implementation of polysaccharide-based vaccines has massively reduced the incidence of invasive pneumococcal diseases. However, there is great concern regarding serotype replacement and the increase in antibiotic resistant strains expressing non-vaccine capsular types. In addition, conjugate vaccines have high production costs, a limiting factor for their implementation in mass immunization programs in developing countries. These limitations have prompted the development of novel vaccine strategies for prevention of Streptococcus pneumoniae infections. The use of conserved pneumococcal antigens such as recombinant proteins or protein fragments presents an interesting serotype-independent alternative. Pht is a family of surface-exposed proteins which have been evaluated as potential vaccine candidates with encouraging results. The present work investigated the immune responses elicited by subcutaneous immunization of mice with the polyhistidine triad protein D (PhtD) and its amino and carboxyl terminal fragments. The proteins were immunogenic and protective against pneumococcal sepsis in mice. Antibodies raised against PhtD increased complement C3b deposition on the pneumococcal surface,mainly mediated by the alternative pathway. Sera from mice immunized with PhtD and PhtD_Cter promoted an increase in bacterial uptake by mouse phagocytes. The interaction of PhtD with the complement system regulator factor H was investigated in silico and in vitro by ELISA and western blot,confirming PhtD as a factor-H binding protein. Our results support the inclusion of PhtD and more specifically, its C-terminal fragment in a multicomponent serotype independent vaccine and suggests a role for the complement system in PhtD-mediated protection. Refereed/Peer-reviewed

In this study, the potential of using the novel antibiotic NCL195 combined with subinhibitory concentrations of colistin against infections caused by Gram-negative bacteria (GNB) was investigated. We showed synergistic activity of the combination NCL195 + colistin against clinical multidrug-resistant GNB pathogens with minimum inhibitory concentrations (MICs) for NCL195 ranging from 0.5–4 μg/mL for Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, whereas NCL195 alone had no activity. Transmission electron microscopy of the membrane morphology of E. coli and P. aeruginosa after single colistin or combination drug treatment showed marked ultrastructural changes most frequently in the cell envelope. Exposure to NCL195 alone did not show any change compared with untreated control cells, whereas treatment with the NCL195 + colistin combination caused more damage than colistin alone. Direct evidence for this interaction was demonstrated by fluorescence-based membrane potential measurements. We conclude that the synergistic antimicrobial activity of the combination NCL195 + colistin against GNB pathogens warrants further exploration for specific treatment of acute GNB infections. Refereed/Peer-reviewed

usc Multidrug-resistant (MDR) pathogens, particularly the ESKAPE group (Enterococcus faecalis/faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, and Enterobacter spp.), have become a public health threat worldwide. Development of new antimicrobial classes and the use of drugs in combination are potential strategies to treat MDR ESKAPE pathogen infections and promote optimal antimicrobial stewardship. Here, the in vitro antimicrobial activity of robenidine analog NCL195 alone or in combination with different concentrations of three outer membrane permeabilizers [ethylenediaminetetraacetic acid (EDTA), polymyxin B nonapeptide (PMBN), and polymyxin B (PMB)] was further evaluated against clinical isolates and reference strains of key Gram-negative bacteria. NCL195 alone was bactericidal against Neisseria meningitidis and Neisseria gonorrhoeae (MIC/MBC = 32 μg/mL) and demonstrated synergistic activity against P. aeruginosa, E. coli, K. pneumoniae, and Enterobacter spp. strains in the presence of subinhibitory concentrations of EDTA, PMBN, or PMB. The additive and/or synergistic effects of NCL195 in combination with EDTA, PMBN, or PMB are promising developments for a new chemical class scaffold to treat Gram-negative infections. Tokuyasu cryo ultramicrotomy was used to visualize the effect of NCL195 on bioluminescent S. aureus membrane morphology. Additionally, NCL195’s favorable pharmacokinetic and pharmacodynamic profile was further explored in in vivo safety studies in mice and preliminary efficacy studies against Gram-positive bacteria. Mice administered two doses of NCL195 (50 mg/kg) by the intraperitoneal (IP) route 4 h apart showed no adverse clinical effects and no observable histological effects in major organs. In bioluminescent Streptococcus pneumoniae and S. aureus murine sepsis challenge models, mice that received two 50 mg/kg doses of NCL195 4 or 6 h apart exhibited significantly reduced bacterial loads and longer survival times than untreated mice. However, further medicinal chemistry and pharmaceutical development to improve potency, solubility, and selectivity is required before efficacy testing in Gram-negative infection models. Refereed/Peer-reviewed

Desymmetrisation of robenidine (1: N ',2-bis((E)-4-chlorobenzylidene)hydrazine-1-carboximidhydrazide) and the introduction of imine alkyl substituents gave good antibiotic activity. Of note was the increased potency of two analogues against vancomycin-resistant Enterococci (VRE), one of which returned a MIC of 0.5 mu g mL(-1). Five analogues were found to be equipotent or more potent than the lead 1. Introduction of an indole moiety resulted in the most active robenidine analogue against methicillin-resistant S. aureus (MRSA), with a MIC of 1.0 mu g mL(-1). Imine C=NH isosteres (C=O/C=S) were inactive. Monomeric analogues were 16-64 mu g mL(-1) active against MRSA and VRE. An analogue that lacks the terminal hydrazide NH moiety showed modest Gram-negative activity at 64 mu g mL(-1). A 4-tert-butyl analogue was shown to be active against both Gram-positive and -negative strains at 16-64 mu g mL(-1). In general, additional modifications with aromatic moieties was poorly tolerated, except with concomitant introduction of an imine C-alkyl group. The activity of these analogues against MRSA and VRE ranged from 8 mu g mL(-1) to inactive (MIC>128 mu g mL(-1)) with the naphthyl and indole analogues. Gram-negative activity was most promising with two compounds at 16 mu g mL(-1) against E. coli. Against P. aeruginosa, the highest activity observed was with MIC values of 32 mu g mL(-1) with another two analogues. Combined, these findings support the further development of the (E)-2-benzylidenehydrazine-1-carboximidamide scaffold as a promising scaffold for the development of antibiotics against Gram-positive and Gram-negative strains. Refereed/Peer-reviewed

The bacterial species and specific spoilage organisms associated with the Southern Australian King George Whiting (KGW) and Tasmanian Atlantic Salmon (TAS), and the efficacy of a HOCl-containing water-based sanitization product (Electro-Chemically Activated Solution, by ECAS4) in extending the shelf life of KGW and TAS fillets were evaluated. Fillets were washed with an ECAS4 solution containing either 45 ppm or 150 ppm of free chlorine and bacterial species enumerated on selective and non-selective media, followed by identification of pure isolates by 16 S rRNA gene sequencing. The dominant spoilage microbiota in KGW and TAS fillets stored at 4 ± 1 °C were Pseudomonas spp. and Shewanella spp. At either concentration, ECAS4 significantly reduced total bacterial load and specific spoilage organisms on KGW and TAS fillets (approx. 1–2 log colony-forming units) during storage and significantly extended the shelf life of the fillets by 2 and 4 days, respectively. The significant increase in shelf life and quality of fillets was corroborated by raw and cooked sensory evaluation. ECAS4 sanitization could have a significant impact on the overall food industry, translating into health and economic benefits through reduction of food spoilage bacteria and potentially, foodborne pathogens without many of the disadvantages of currently approved biocides Refereed/Peer-reviewed

Streptococcus pneumoniae (the pneumococcus) is a human pathogen, accounting for massive global morbidity and mortality. Although asymptomatic colonization of the nasopharynx almost invariably precedes disease, the critical determinants enabling pneumococcal progression from this niche to cause invasive disease are poorly understood. One mechanism proposed to be central to this transition involves opacity phase variation, whereby pneumococci harvested from the nasopharynx are typically transparent, while those simultaneously harvested from the blood are opaque. Here, we used two dimensional-differential gel electrophoresis (2D-DIGE) to compare protein expression profiles of transparent and opaque variants of 3 pneumococcal strains, D39 (serotype 2), WCH43 (serotype 4) and WCH16 (serotype 6A) in vitro. One spot comprising a mixture of capsular polysaccharide biosynthesis protein and other proteins was significantly up-regulated in the opaque phenotype in all 3 strains; other proteins were differentially regulated in a strain-specific manner. We conclude that pneumococcal phase variation is a complex and multifactorial process leading to strain-specific pathogenicity.

There is increasing demand for safe and effective sanitizers for irrigation water disinfection to prevent transmission of foodborne pathogens to fresh produce. Here we compared the efficacy of pH-neutral electrolyzed oxidizing water (EOW), sodium hypochlorite (NaClO) and chlorine dioxide (ClO2) against single and mixed populations of E. coli, Listeria and Salmonella under a range of pH and organic matter content. EOW treatment of the mixed bacterial suspension resulted in a dose-dependent (10) reduction of the microbial load in water devoid of organic matter under the range of pH conditions tested (pH, 6.0, 7.0, 8.4 and 9.2). The efficacy of EOW containing 5 mg/L free chlorine was unaffected by increasing organic matter, and compared favourably with equivalent concentrations of NaClO and ClO2. EOW at 20 mg/L free chlorine was more effective than NaClO and ClO2 in reducing bacterial populations in the presence of high (20–100 mg/L) dissolved organic carbon, and no regrowth or metabolic activity was observed for EOW-treated bacteria at this concentration upon reculturing in rich media. Thus, EOW is as effective or more effective than other common chlorine-based sanitizers for pathogen reduction in contaminated water. EOW’s other characteristics, such as neutral pH and ease of handling, indicate its suitability for fresh produce sanitation.

Stone formation and catheter blockage are major complications of Proteus UTIs. In this study, we investigated the ability of allicin to inhibit P. mirabilis-induced struvite crystallization and catheter blockage using a synthetic bladder model. Struvite crystallization inhibition study was carried out using P. mirabilis lysate as urease enzyme source in synthetic urine (SU). Struvite productions were monitored by phase contrast light microscopy and measurements of pH, Mg 2+ and Ca 2+ precipitation and turbidity. A catheter blockage study was performed in a synthetic bladder model mimicking natural UTI in the presence of allicin at sub-MIC concentrations (MIC = 64 μg/ml). The results of crystallization study showed that allicin inhibited pH rise and consequently turbidity and precipitation of ions in a dose-dependent manner. The results of catheter blockage study showed that allicin at sub-MIC concentrations (2, 4, 8 μg/ml) significantly increased the time for catheter blockage to occur to 61, 74 and 92 h respectively compared to allicin-free control (48 h). In a similar way, the results showed that allicin delayed the increase of SU pH level in bladder model in a dose-dependent manner compared to allicin-free control. The results also showed that following the increase of allicin concentration, Mg 2+ and Ca 2+ deposition in catheters were much lower compared to allicin-free control, further confirmed by direct observation of the catheters’ eyehole and cross sections. We conclude that allicin prevents the formation of Proteus-induced urinary crystals and the blockage of catheters by delaying pH increase and lowering Mg 2+ and Ca 2+ deposition in a dose-dependent manner. Refereed/Peer-reviewed

The emergence and global spread of antimicrobial resistance among bacterial pathogens demand alternative strategies to treat life-threatening infections. Combination drugs and repurposing of old compounds with known safety profiles that are not currently used in human medicine can address the problem of multidrug-resistant infections and promote antimicrobial stewardship in veterinary medicine. In this study, the antimicrobial activity of robenidine alone or in combination with ethylenediaminetetraacetic acid (EDTA) or polymyxin B nonapeptide (PMBN) against Gram-negative bacterial pathogens, including those associated with canine otitis externa and human skin and soft tissue infection, was evaluated in vitro using microdilution susceptibility testing and the checkerboard method. Fractional inhibitory concentration indices (FICIs) and dose reduction indices (DRI) of the combinations against tested isolates were determined. Robenidine alone was bactericidal against Acinetobacter baumannii [minimum inhibitory concentrations (MIC) mode = 8 μg/ml] and Acinetobacter calcoaceticus (MIC mode = 2 μg/ml). Against Acinetobacter spp., an additivity/indifference of the combination of robenidine/EDTA (0.53 > FICIs > 1.06) and a synergistic effect of the combination of robenidine/PMBN (0.5 < FICI) were obtained. DRIs of robenidine were significantly increased in the presence of both EDTA and PMBN from 2- to 2048-fold. Robenidine exhibited antimicrobial activity against Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, in the presence of sub-inhibitory concentrations of either EDTA or PMBN. Robenidine also demonstrated potent antibacterial activity against multidrug-resistant Gram-positive pathogens and all Gram-negative pathogens isolated from cases of canine otitis externa in the presence of EDTA. Robenidine did not demonstrate antibiofilm activity against Gram-positive and Gram-negative bacteria. EDTA facilitated biofilm biomass degradation for both Gram-positives and Gram-negatives. The addition of robenidine to EDTA was not associated with any change in the effect on biofilm biomass degradation. The combination of robenidine with EDTA or PMBN has potential for further exploration and pharmaceutical development, such as incorporation into topical and otic formulations for animal and human use. Refereed/Peer-reviewed

An electrochemically activated solution (ECAS), generated by electrolysis of a dilute sodium chloride solution in a four-chamber electrolytic cell (Ecas4), was tested as a sanitising aerosol in eliminating bacteria from the environment of a weaning room vacated 24-48h earlier, at a continuous flow pig farm. An ultrasonic humidifier was used to fill the environment with a fog (droplets with diameters of 1–5 μm) containing 0.25 ppm of hypochlorous acid. The weaning room was fogged for 3 min at 30 min intervals during five hours of aerosol disinfection. An innovative sample treatment with propidium monoazide dye in conjunction with cyclonic air sampling was optimised and adapted for discerning live/dead bacteria in subsequent molecular quantification steps. Without fogging, total bacterial load ranged from 5.06 ± 0.04 to 5.75 ± 0.04 Log10 CFU/m3. After the first hour of fogging, a 78% total bacterial reduction was observed, which further increased to > 97% after the second hour, > 99.4% after the third and 99.8% after the fourth hour, finally resulting in a 99.99% reduction from the farm environment over five hours. Unlike the current formaldehyde spray disinfection protocol, which requires a long empty period because of its hazardous properties, this economically viable and environmentally friendly disinfection protocol may significantly lower downtime. Moreover, ECAS fogging can be easily adapted to a variety of applications, including the elimination of pathogens from livestock farm air environment for disease prevention, as well as decontamination after disease outbreaks. Refereed/Peer-reviewed

There are growing demands globally to use safe, efficacious and environmentally friendly sanitizers for post-harvest treatment of fresh produce to reduce or eliminate spoilage and foodborne pathogens. Here, we compared the efficacy of a pH-neutral electrolyzed oxidizing water (Ecas4 Anolyte; ECAS) with that of an approved peroxyacetic acid-based sanitizer (Ecolab Tsunami® 100) in reducing the total microbial load and inoculated Escherichia coli, Salmonella Enteritidis and Listeria innocua populations on post-harvest baby spinach leaves over 10 days. The impact of both sanitizers on the overall quality of the spinach leaves during storage was also assessed by shelf life and vitamin C content measurements. We show that ECAS at 50 ppm and 85 ppm significantly reduced the bacterial load compared to tap water-treated or untreated (control) leaves, and at similar levels (approx. 10-fold reduction) to those achieved using 50 ppm of Ecolab Tsunami®. While there were no obvious deleterious effects of treatment with 50 ppm Tsunami® or ECAS at 50 ppm and 85 ppm on plant leaf appearance, tap water-treated and untreated leaves showed some yellowing, bruising and sliming. Given its safety, efficacy and environmentally-friendly characteristics, ECAS could be a viable alternative to chemical-based sanitizers for post-harvest treatment of fresh produce. Refereed/Peer-reviewed

The bacterial cell division protein, FtsZ, has been identified as a target for antimicrobial development. Derivatives of 3-methoxybenzamide have shown promising activities as FtsZ inhibitors in Gram-positive bacteria. We sought to characterise the activity of five difluorobenzamide derivatives with non-heterocyclic substituents attached through the 3-oxygen. These compounds exhibited antimicrobial activity against methicillin resistant Staphylococcus aureus (MRSA), with an isopentyloxy-substituted compound showing modest activity against vancomycin resistant Enterococcus faecium (VRE). The compounds were able to reverse resistance to oxacillin in highly resistant clinical MRSA strains at concentrations far below their MICs. Three of the compounds inhibited an Escherichia coli strain lacking the AcrAB components of a drug efflux pump, which suggests the lack of Gram-negative activity can partly be attributed to efflux. The compounds inhibited cell division by targeting S. aureus FtsZ, producing a dose-dependent increase in GTPase rate which increased the rate of FtsZ polymerization and stabilized the FtsZ polymers. These compounds did not affect the polymerization of mammalian tubulin and did not display haemolytic activity or cytotoxicity. These derivatives are therefore promising compounds for further development as antimicrobial agents or as resistance breakers to re-sensitive MRSA to beta-lactam antibiotics.

Ready-to-eat (RTE) spinach is considered a high-risk food, susceptible to colonization by foodborne pathogens, however, other microbial populations present on the vegetable surface may interact with foodborne pathogens by inhibiting/inactivating their growth. In addition, sanitizers applied to minimally processed salad leaves should not disrupt this autochthonous barrier and should be maintained throughout the shelf life of the product. This investigation aimed at comparing the effects of a pH neutral electrochemically activated solution (ECAS), a peroxyacetic acid (PAA)-based commercial sanitizer (Ecolab Tsunami&reg, 100), and tap water wash on the minimally processed spinach leaf microbiome profile for 10 days after washing. The bacterial microbiota composition on spinach samples was assessed by 16S rRNA pyrosequencing and downstream analyses. Predominant phyla observed in decreasing order of abundance were Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes corresponding with the dominant families Micrococcaceae, Clostridiales Family XII, Flavobacteriaceae, Pseudomonadaceae, and Burkholderiaceae. Bacterial species richness and evenness (alpha diversity) and bacterial community composition among all wash types were not significantly different. However, a significant difference was apparent between sampling days, corresponding to a loss of overall heterogeneity over time. Analysis of composition of microbiome (ANCOM) did not identify any amplicon sequence variants (ASVs) or families having significantly different abundance in wash types, however, differences (17 ASVs and five families) were found depending on sampling day. This was the first bacterial microbiome composition study focused on ECAS and PAA-based wash solutions. These wash alternatives do not significantly alter microbial community composition of RTE spinach leaves, however, storage at refrigerated temperature reduces bacterial species heterogeneity.

Increasing reports of multidrug-resistant bacterial infections in animals has created a need for novel antimicrobial agents that do not promote cross-resistance to critically important antimicrobial classes used in human medicine. In response to the recent emergence of antimicrobial resistance in several bovine mastitis pathogens, in vitro antimicrobial susceptibility was determined for four polyether ionophores (lasalocid, monensin, narasin and salinomycin) against Staphylococcus spp. and Streptococcus spp. isolated from clinical cases. In addition, erythrocyte haemolysis and WST-1 cell proliferation assays were used to assess in vitro mammalian cell cytotoxicity and biofilm susceptibility testing was performed using the minimum biofilm eradication concentration(MBEC™) biofilm assay. Lasalocid, monensin, narasin and salinomycin exhibited bacteriostatic antimicrobial activity against all pathogens tested, including methicillin-resistant staphylococci, with MIC90 values

There are very few articles in the literature describing continuous models of bacterial infections that mimic disease pathogenesis in humans and animals without using separate cohorts of animals at each stage of disease. In this work, we developed bioluminescent mouse models of partial-thickness scald wound infection and sepsis that mimic disease pathogenesis in humans and animals using a recombinant luciferase-expressing Staphylococcus aureus strain (Xen29). Two days post-scald wound infection, mice were treated twice daily with a 2% topical mupirocin ointment for 7 days. For sepsis experiments, mice were treated intraperitoneally with 6 mg/kg daptomycin 2 h and 6 h post-infection and time to moribund monitored for 72 h. Consistent bacterial burden data were obtained from individual mice by regular photon intensity quantification on a Xenogen IVIS Lumina XRMS Series III biophotonic imaging system, with concomitant significant reduction in photon intensities in drug-treated mice. Post-mortem histopathological examination of wounds and bacterial counts in blood correlated closely with disease severity and total flux obtained from Xen29. The bioluminescent murine models provide a refinement to existing techniques of multiple bacterial enumeration during disease pathogenesis and promote animal usage reduction. The models also provide an efficient and information-rich platform for preclinical efficacy evaluation of new drug classes for treating acute and chronic human and animal bacterial infections. Refereed/Peer-reviewed

Methicillin-resistant coagulase-negative staphylococci (MRCoNS) have recently emerged as a significant cause of bovine mastitis worldwide. Here we describe the isolation of MRCoNS from cases of bovine mastitis from a single dairy farm in Australia. Fourteen CoNS isolates were identified as MRCoNS on the basis of having an oxacillin MIC of ≥0.5 μg/mL. The isolates were speciated as S. chromogenes (n = 1) S. fleurettii (n = 1), S. haemolyticus (n = 2), S. sciuri (n = 5), S. simulans (n = 1) S. succinus (n = 2) and S. xylosus (n = 2). Five of the isolates (S. fleuretti, S. haemolyticus S. sciuri and two S. succinus) were mecA-positive. We also detected a previously described S. sciuri mecA homolog in four oxacillin-resistant S. sciuri isolates. The remainder of the putative MRCoNS did not contain any mecA-related resistance determinants in their genomes. Comparative genomic analysis of three previously published S. sciuri isolates, from humans, a squirrel and a cereal crop (rice), and a representative isolate from our study demonstrated clustering and a high degree of genetic homogeneity ( > 95%), suggesting S. sciuri has low host specificity. In conclusion, CoNS, in particular S. sciuri, may act as a reservoir for SCCmec elements that can easily be spread between different host species by direct cross-infection. Refereed/Peer-reviewed

Introduction : it is projected that by 2050, 40% of 10 million deaths from Antimicrobial Resistance (AMR) will occur in Africa. Understanding the AMR situation in Nigeria will provide an excellent case study of the challenges faced by low-income countries. Methods : the information was derived from review of reports, programmatic data and documents, literature search, key informant interviews and a series of systematic reviews. Data was entered into purpose-built templates and synthesized thematically. Results : in Nigeria, the ratio of licensed pharmacies to over-the-counter medicine stores was 15 to 1 in 2016. A systematic review determined that median prevalence of persons using antibiotics without prescription to be 46.8%. In animals, antibiotics such as tetracyclines constituted over 80% of antimicrobials sold or used in 2014 and 2015. Antibiotic resistance was documented in humans, to drugs recommended by the country’s treatment guidelines for commonly occurring infections such as cholera and cerebrospinal meningitis. Majority of the studies documented recovery E. coli, non-typhoidal Salmonella and antibiotic residues from livestock, pets and animal products, most commonly in poultry. The drivers of AMR included unregulated antibiotic sales, proliferation of unlicensed medicine stores, shortage of licensed prescribers, poor AMR awareness and use of antibiotics in animals without prescription. Conclusion : we recommend that the government enforce regulations on antibiotic sales of antibiotics to humans and animals and increase awareness on AMR in Nigerian communities. Identified gaps from the situation analysis were used to develop a National Action Plan for AMR.

Made available in DSpace on 2018-11-26T17:40:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-09-05 Australian Research Council (ARC) Neoculi Pty Ltd National Health and Medical Research Council of Australia (NHMRC) University of South Australia Sansom Institute of Health Research Micromyx LLC The spread of multidrug resistance among bacterial pathogens poses a serious threat to public health worldwide. Recent approaches towards combating antimicrobial resistance include repurposing old compounds with known safety and development pathways as new antibacterial classes with novel mechanisms of action. Here we show that an analog of the anticoccidial drug robenidine (4,6-bis(2-((E)-4-methylbenzylidene)hydrazinyl)pyrimidin-2-amine; NCL195) displays potent bactericidal activity against Streptococcus pneumoniae and Staphylococcus aureus by disrupting the cell membrane potential. NCL195 was less cytotoxic to mammalian cell lines than the parent compound, showed low metabolic degradation rates by human and mouse liver microsomes, and exhibited high plasma concentration and low plasma clearance rates in mice. NCL195 was bactericidal against Acinetobacter spp and Neisseria meningitidis and also demonstrated potent activity against A. baumannii, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and Enterobacter spp. in the presence of sub-inhibitory concentrations of ethylenediaminetetraacetic acid (EDTA) and polymyxin B. These findings demonstrate that NCL195 represents a new chemical lead for further medicinal chemistry and pharmaceutical development to enhance potency, solubility and selectivity against serious bacterial pathogens. Univ Adelaide, Sch Anim & Vet Sci, Australian Ctr Antimicrobial Resistance Ecol, Roseworthy, SA, Australia Univ Newcastle, Sch Environm & Life Sci, Chem, Callaghan, NSW, Australia Univ Adelaide, Sch Anim & Vet Sci, Roseworthy, SA, Australia Neoculi Pty Ltd, Burwood, Vic, Australia Univ South Australia, Sansom Inst Hlth Res, Sch Pharm & Med Sci, Ctr Pharmaceut Innovat & Dev, Adelaide, SA, Australia Univ South Australia, Sansom Inst Hlth Res, Sch Pharm & Med Sci, Adelaide, SA, Australia Monash Univ, Monash Inst Pharmaceut Sci, Ctr Drug Candidate Optimisat, Parkville, Vic, Australia Sao Paulo State Univ, Sch Pharmaceut Sci, Dept Drugs & Med, Sao Paulo, Brazil Fiona Stanley Hosp, Path West Lab Med WA, Dept Microbiol, Murdoch, WA, Australia Murdoch Univ, Sch Vet & Life Sci, Murdoch, WA, Australia Micromyx LLC, Kalamazoo, MI USA Univ Adelaide, Sch Biol Sci, Dept Mol & Cellular Biol, Adelaide, SA, Australia Univ Adelaide, Sch Biol Sci, Dept Mol & Cellular Biol, Res Ctr Infect Dis, Adelaide, SA, Australia Sao Paulo State Univ, Sch Pharmaceut Sci, Dept Drugs & Med, Sao Paulo, Brazil Australian Research Council (ARC): LP110200770 National Health and Medical Research Council of Australia (NHMRC): 565526 National Health and Medical Research Council of Australia (NHMRC): 627142

Drug efflux pumps confer multidrug resistance to dangerous pathogens which makes these pumps important drug targets. We have synthesised a novel series of compounds based on a 2-naphthamide pharmacore aimed at inhibiting the efflux pumps from Gram-negative bacteria. The archeatypical transporter AcrB from Escherichia coli was used as model efflux pump as AcrB is widely conserved throughout Gram-negative organisms. The compounds were tested for their antibacterial action, ability to potentiate the action of antibiotics and for their ability to inhibit Nile Red efflux by AcrB. None of the compounds were antimicrobial against E. coli wild type cells. Most of the compounds were able to inhibit Nile Red efflux indicating that they are substrates of the AcrB efflux pump. Three compounds were able to synergise with antibiotics and reverse resistance in the resistant phenotype. Compound A3, 4-(isopentyloxy)-2-naphthamide, reduced the MICs of erythromycin and chloramphenicol to the MIC levels of the drug sensitive strain that lacks an efflux pump. A3 had no effect on the MIC of the non-substrate rifampicin indicating that this compound acts specifically through the AcrB efflux pump. A3 also does not act through non-specific mechanisms such as outer membrane or inner membrane permeabilisation and is not cytotoxic against mammalian cell lines. Therefore, we have designed and synthesised a novel chemical compound with great potential to further optimisation as inhibitor of drug efflux pumps. Refereed/Peer-reviewed

Streptococcus pneumoniae (the pneumococcus) is a major human pathogen, causing a broad spectrum of diseases including otitis media, pneumonia, bacteraemia and meningitis. Here we examined the role of a potential pneumococcal meningitis vaccine antigen, alpha-glycerophosphate oxidase (SpGlpO), in nasopharyngeal colonization. We found that serotype 4 and serotype 6A strains deficient in SpGlpO have significantly reduced capacity to colonize the nasopharynx of mice, and were significantly defective in adherence to human nasopharyngeal carcinoma cells in vitro. We also demonstrate that intranasal immunization with recombinant SpGlpO significantly protects mice against subsequent nasal colonization by wild type serotype 4 and serotype 6A strains. Furthermore, we show that SpGlpO binds strongly to lacto/neolacto/ganglio host glycan structures containing the GlcNAcβ1-3Galβ disaccharide, suggesting that SpGlpO enhances colonization of the nasopharynx through its binding to host glycoconjugates. We propose that SpGlpO is a promising vaccine candidate against pneumococcal carriage, and warrants inclusion in a multi-component protein vaccine formulation that can provide robust, serotype-independent protection against all forms of pneumococcal disease., Highlights • Streptococcus pneumoniae GlpO (SpGlpO) enhances nasal colonization of mice through binding to specific host glycoconjugates. • Mutants deficient in SpGlpO colonize the mouse nasopharynx poorly. • Nasal immunization with SpGlpO protects mice against S. pneumoniae colonization. New vaccines that protect against nasal colonization and all forms of diseases caused by Streptococcus pneumoniae (the pneumococcus) are urgently needed. In this manuscript, Mahdi et al. show that alpha-glycerophosphate oxidase (SpGlpO), a protein present in all pneumococci, enhances nasal colonization of mice through binding to specific host glycoconjugates, and strains lacking SpGlpO colonize the mouse nasopharynx poorly. Intranasal immunization with SpGlpO significantly protects mice against subsequent S. pneumoniae colonization, indicating SpGlpO is a promising protein vaccine candidate against pneumococcal carriage.

Robenidine, 1 (2,2′-bis[(4-chlorophenyl)methylene]carbonimidic dihydrazide), was active against MRSA and VRE with MIC’s of 8.1 and 4.7 μM, respectively. SAR revealed tolerance for 4-Cl isosteres with 4-F (8), 3-F (9), 3-CH3 (22), and 4-C(CH3)3 (27) (23.7–71 μM) and with 3-Cl (3), 4-CH3 (21), and 4-CH(CH3)2 (26) (8.1–13.0 μM). Imine carbon alkylation identified a methyl/ethyl binding pocket that also accommodated a CH2OH moiety (75; 2,2′-bis[1-(4-chlorophenyl)-2-hydroxyethylidene]carbonimidic dihydrazide). Analogues 1, 27 (2,2′-bis{[4-(1,1-dimethylethyl)phenyl]methylene}carbonimidic dihydrazide), and 69 (2,2′-bis[1-(4-chlorophenyl)ethylidene]carbonimidic dihydrazide hydrochloride) were active against 24 clinical MRSA and MSSA isolates. No dose-limiting cytotoxicity at ≥2× MIC or hemolysis at ≥8× MIC was observed. Polymyxin B addition engendered Escherichia coli and Pseudomonas aeruginosa Gram-negative activity MIC’s of 4.2–21.6 μM. 1 and 75 displayed excellent microsomal stability, intrinsic clearance, and hepatic extraction ratios with T1/2 > 247 min, CLint < 7 μL/min/mg protein, and EH < 0.22 in both human and mouse liposomes for 1 and in human liposomes for 75. Refereed/Peer-reviewed

Generating a pneumococcal vaccine that is serotype independent and cost effective remains a global challenge. γ-Irradiation has been used widely to sterilize biological products. It can also be utilized as an inactivation technique to generate whole-cell bacterial and viral vaccines with minimal impact on pathogen structure and antigenic determinants. In the present study, we utilized γ-irradiation to inactivate an un-encapsulated Streptococcus pneumoniae strain Rx1 with an unmarked deletion of the autolysin gene lytA and with the pneumolysin gene ply replaced with an allele encoding a non-toxic pneumolysoid (PdT) (designated γ-PN vaccine). Intranasal vaccination of C57BL/6 mice with γ-PN was shown to elicit serotype-independent protection in lethal challenge models of pneumococcal pneumonia and sepsis. Vaccine efficacy was shown to be reliant on B-cells and interleukin (IL)-17A responses. Interestingly, immunization promoted IL-17 production by innate cells not T helper 17 (Th17) cells. These data are the first to report the development of a non-adjuvanted intranasal γ-irradiated pneumococcal vaccine that generates effective serotype-independent protection, which is mediated by both humoral and innate IL-17 responses. Refereed/Peer-reviewed

Streptococcus pneumoniae (the pneumococcus) continues to account for significant morbidity and mortality worldwide, causing life-threatening diseases such as pneumonia, bacteremia and meningitis, as well as less serious infections such as sinusitis, conjunctivitis and otitis media. Current polysaccharide vaccines are strictly serotype-specific and also drive the emergence of non-vaccine serotype strains. In this study, we used microarray analysis to compare gene expression patterns of either serotype 4 or serotype 6A pneumococci in the nasopharynx and blood of mice, as a model to identify genes involved in invasion of blood in the context of occult bacteremia in humans. In this manner, we identified 26 genes that were significantly up-regulated in the nasopharynx and 36 genes that were significantly up-regulated in the blood that were common to both strains. Gene Ontology classification revealed that transporter and DNA binding (transcription factor) activities constitute the significantly different molecular functional categories for genes up-regulated in the nasopharynx and blood. Targeted mutagenesis of selected genes from both niches and subsequent virulence and pathogenesis studies identified the manganese-dependent superoxide dismutase (SodA) as most likely to be essential for colonization, and the cell wall-associated serine protease (PrtA) as important for invasion of blood. This work extends our previous analyses and suggests that both PrtA and SodA warrant examination in future studies aimed at prevention and/or control of pneumococcal disease. Refereed/Peer-reviewed

IL-17-producing helper T (Th17) cells are critical for host defense against extracellular pathogens but also drive numerous autoimmune diseases. Th17 cells that differ in their inflammatory potential have been described including IL-10-producing Th17 cells that are weak inducers of inflammation and highly inflammatory, IL-23-driven, GM-CSF/IFNγ-producing Th17 cells. However, their distinct developmental requirements, functions and trafficking mechanisms in vivo remain poorly understood. Here we identify a temporally regulated IL-23-dependent switch from CCR6 to CCR2 usage by developing Th17 cells that is critical for pathogenic Th17 cell-driven inflammation in experimental autoimmune encephalomyelitis (EAE). This switch defines a unique in vivo cell surface signature (CCR6−CCR2+) of GM-CSF/IFNγ-producing Th17 cells in EAE and experimental persistent extracellular bacterial infection, and in humans. Using this signature, we identify an IL-23/IL-1/IFNγ/TNFα/T-bet/Eomesodermin-driven circuit driving GM-CSF/IFNγ-producing Th17 cell formation in vivo. Thus, our data identify a unique cell surface signature, trafficking mechanism and T-cell intrinsic regulators of GM-CSF/IFNγ-producing Th17 cells., Little is known regarding migration of Th17 cells that produce distinct cytokines implicated in protection and pathology. Kara et al. show that a switch from CCR6 to CCR2 by Th17 cells defines a signature (CCR6−CCR2+) of GM-CSF+ Th17 cells and drives pathology in a mouse model of autoimmunity.

Streptococcus pneumoniae is a leading cause of mortality in young children. While successful conjugate polysaccharide vaccines exist, a less expensive serotype-independent protein-based pneumococcal vaccine offers a major advancement for preventing life-threatening pneumococcal infections, particularly in developing nations. IL-17A-secreting CD4+ T cells (TH17) mediate resistance to mucosal colonization by multiple pathogens including S. pneumoniae. Screening an expression library containing >96% of predicted pneumococcal proteins, we identified antigens recognized by TH17 cells from mice immune to pneumococcal colonization. The identified antigens also elicited IL-17A secretion from colonized mouse splenocytes and human PBMCs suggesting that similar responses are primed during natural exposure. Immunization of two mouse strains with identified antigens provided protection from pneumococcal colonization that was significantly diminished in animals treated with blocking CD4 or IL-17A antibodies. This work demonstrates the potential of proteomic screening approaches to identify specific antigens for the design of subunit vaccines against mucosal pathogens via harnessing TH17-mediated immunity.

Background Streptococcus pneumoniae (the pneumococcus) is the world’s foremost microbial pathogen, killing more people each year than HIV, TB or malaria. The capacity to penetrate deeper host tissues contributes substantially to the ability of this organism to cause disease. Here we investigated, for the first time, functional genomics modulation of 3 pneumococcal strains (serotype 2 [D39], serotype 4 [WCH43] and serotype 6A [WCH16]) during transition from the nasopharynx to lungs to blood and to brain of mice at both promoter and domain activation levels. Results We found 7 highly activated transcription factors (TFs) [argR, codY, hup, rpoD, rr02, scrR and smrC] capable of binding to a large number of up-regulated genes, potentially constituting the regulatory backbone of pneumococcal pathogenesis. Strain D39 showed a distinct profile in employing a large number of TFs during blood infection. Interestingly, the same highly activated TFs used by D39 in blood are also used by WCH16 and WCH43 during brain infection. This indicates that different pneumococcal strains might activate a similar set of TFs and regulatory elements depending on the final site of infection. Hierarchical clustering analysis showed that all the highly activated TFs, except rpoD, clustered together with a high level of similarity in all 3 strains, which might suggest redundancy in the regulatory roles of these TFs during infection. Discriminant function analysis of the TFs in various niches highlights differential regulatory backgrounds of the 3 strains, and pathogenesis data confirms codY as the most significant predictor discriminating between these strains in various niches, particularly in the blood. Moreover, the predicted TF and domain activation profiles of the 3 strains correspond with their distinct pathogenicity characteristics. Conclusions Our findings suggest that the pneumococcus changes the short binding sites in the promoter regions of genes in a niche-specific manner to enhance its ability to disseminate from one host niche to another. This study provides a framework for an improved understanding of the dynamics of pneumococcal pathogenesis, and opens a new avenue into similar investigations in other pathogenic bacteria. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-769) contains supplementary material, which is available to authorized users.

Polyhistidine triad protein D (PhtD) has been described as a promising vaccine candidate for use against Streptococcus pneumoniae, but there has been a lack of examination of its structure and of which region(s) of the protein are targeted by protective immune responses. In this study, we purified recombinant truncated derivatives of PhtD and examined their secondary structural composition, as well as their capacity to bind antibodies from polyclonal murine serum generated against the full length protein. This allowed the identification of a particularly immunogenic fragment of PhtD, which was also purified and characterised. The truncated derivatives were tested as vaccine antigens in mouse models of pneumococcal sepsis and colonisation, using alum and E. coli heat labile toxin B subunit respectively as adjuvants. These experiments revealed that whilst the immunogenic region identified may be a promising candidate to protect against sepsis, the full length PhtD was ineffective at conferring significant protective immunity. These results are significant for the potential for PhtD to be used in novel vaccines, which are currently being tested in clinical trials. Refereed/Peer-reviewed

To date, the role of transcription factors (TFs) in the progression of disease for many pathogens is yet to be studied in detail. This is probably due to transient, and generally low expression levels of TFs, which are the central components controlling the expression of many genes during the course of infection. However, a small change in the expression or specificity of a TF can radically alter gene expression. In this study, we combined a number of quality-based selection strategies including structural prediction of modulated genes, gene ontology and network analysis, to predict the regulatory mechanisms underlying pathogenesis of Streptococcus pneumoniae (the pneumococcus). We have identified two TFs (SP_0676 and SP_0927 [SmrC]) that might control tissue-specific gene expression during pneumococcal translocation from the nasopharynx to lungs, to blood and then to brain of mice. Targeted mutagenesis and mouse models of infection confirmed the role of SP_0927 in pathogenesis and virulence, and suggests that SP_0676 might be essential to pneumococcal viability. These findings provide fundamental new insights into virulence gene expression and regulation during pathogenesis. Refereed/Peer-reviewed

Pneumococcal disease is associated with a particularly high morbidity and mortality amongst adults in HIV endemic countries. Our previous findings implicating a B-cell defect in HIV-infected children from the same population led us to comprehensively characterize B-cell subsets in minimally symptomatic HIV-infected Malawian adults and investigate the isotype-switched IgG memory B-cell immune response to the pneumococcus. We show that similar to vertically acquired HIV-infected Malawian children, horizontally acquired HIV infection in these adults is associated with IgM memory B-cell (CD19(+) CD27(+) IgM(+) IgD(+)) depletion, B-cell activation and impairment of specific IgG B-cell memory to a range of pneumococcal proteins. Our data suggest that HIV infection affects both T-cell independent and T-cell dependent B-cell maturation, potentially leading to impairment of humoral responses to extracellular pathogens such as the pneumococcus, and thus leaving this population susceptible to invasive disease. Refereed/Peer-reviewed

During infection, pneumococci exist mainly in sessile biofilms rather than in planktonic form, except during sepsis. However, relatively little is known about how biofilms contribute to pneumococcal pathogenesis. Here, we carried out a biofilm assay on opaque and transparent variants of a clinical serotype 19F strain WCH159. After 4 days incubation, scanning electron microscopy revealed that opaque biofilm bacteria produced an extracellular matrix, whereas the transparent variant did not. The opaque biofilm-derived bacteria translocated from the nasopharynx to the lungs and brain of mice, and showed 100-fold greater in vitro adherence to A549 cells than transparent bacteria. Microarray analysis of planktonic and sessile bacteria from transparent and opaque variants showed differential gene expression in two operons: the lic operon, which is involved in choline uptake, and in the two-component system, ciaRH. Mutants of these genes did not form an extracellular matrix, could not translocate from the nasopharynx to the lungs or the brain, and adhered poorly to A549 cells. We conclude that only the opaque phenotype is able to form extracellular matrix, and that the lic operon and ciaRH contribute to this process. We propose that during infection, extracellular matrix formation enhances the ability of pneumococci to cause invasive disease. Refereed/Peer-reviewed

The bacterial factors responsible for the variation in invasive potential between different clones and serotypes of Streptococcus pneumoniae are largely unknown. Therefore, the isolation of rare serotype 1 carriage strains in Indigenous Australian communities provided a unique opportunity to compare the genomes of non-invasive and invasive isolates of the same serotype in order to identify such factors. The human virulence status of non-invasive, intermediately virulent and highly virulent serotype 1 isolates was reflected in mice and showed that whilst both human non-invasive and highly virulent isolates were able to colonize the murine nasopharynx equally, only the human highly virulent isolates were able to invade and survive in the murine lungs and blood. Genomic sequencing comparisons between these isolates identified 8 regions >1 kb in size that were specific to only the highly virulent isolates, and included a version of the pneumococcal pathogenicity island 1 variable region (PPI-1v), phage-associated adherence factors, transporters and metabolic enzymes. In particular, a phage-associated endolysin, a putative iron/lead permease and an operon within PPI-1v exhibited niche-specific changes in expression that suggest important roles for these genes in the lungs and blood. Moreover, in vivo competition between pneumococci carrying PPI-1v derivatives representing the two identified versions of the region showed that the version of PPI-1v in the highly virulent isolates was more competitive than the version from the less virulent isolates in the nasopharyngeal tissue, blood and lungs. This study is the first to perform genomic comparisons between serotype 1 isolates with distinct virulence profiles that correlate between mice and humans, and has highlighted the important role that hypervariable genomic loci, such as PPI-1v, play in pneumococcal disease. The findings of this study have important implications for understanding the processes that drive progression from colonization to invasive disease and will help direct the development of novel therapeutic strategies. Refereed/Peer-reviewed

The primary means of classifying new functions for genes and proteins relies on Gene Ontology (GO), which defines genes/proteins using a controlled vocabulary in terms of their Molecular Function, Biological Process and Cellular Component. The challenge is to present this information to researchers to compare and discover patterns in multiple datasets using visually comprehensible and user-friendly statistical reports. Importantly, while there are many GO resources available for eukaryotes, there are none suitable for simultaneous, graphical and statistical comparison between multiple datasets. In addition, none of them supports comprehensive resources for bacteria. By using Streptococcus pneumoniae as a model, we identified and collected GO resources including genes, proteins, taxonomy and GO relationships from NCBI, UniProt and GO organisations. Then, we designed database tables in PostgreSQL database server and developed a Java application to extract data from source files and loaded into database automatically. We developed a PHP web application based on Model-View-Control architecture, used a specific data structure as well as current and novel algorithms to estimate GO graphs parameters. We designed different navigation and visualization methods on the graphs and integrated these into graphical reports. This tool is particularly significant when comparing GO groups between multiple samples (including those of pathogenic bacteria) from different sources simultaneously. Comparing GO protein distribution among up- or down-regulated genes from different samples can improve understanding of biological pathways, and mechanism(s) of infection. It can also aid in the discovery of genes associated with specific function(s) for investigation as a novel vaccine or therapeutic targets. Availability http://turing.ersa.edu.au/BacteriaGO.

14 p.-1 tab.-8 fig. Shilpa Basavanna et alt., Bacterial pathogens need to acquire nutrients from the host, but for many nutrients their importance during infection remain poorly understood. We have investigated the importance of methionine acquisition and synthesis for Streptococcus pneumoniae growth and virulence using strains with gene deletions affecting a putative methionine ABC transporter lipoprotein (Sp_0149, metQ) and/or methionine biosynthesis enzymes (Sp_0585 - Sp_0586, metE and metF). Immunoblot analysis confirmed MetQ was a lipoprotein and present in all S. pneumoniae strains investigated. However, vaccination with MetQ did not prevent fatal S. pneumoniae infection in mice despite stimulating a strong specific IgG response. Tryptophan fluorescence spectroscopy and isothermal titration calorimetry demonstrated that MetQ has both a high affinity and specificity for L-methionine with a KD of ,25 nM, and a DmetQ strain had reduced uptake of C14-methionine. Growth of the DmetQ/DmetEF strain was greatly impaired in chemically defined medium containing low concentrations of methionine and in blood but was partially restored by addition of high concentrations of exogenous methionine. Mixed infection models showed no attenuation of the DmetQ, DmetEF and DmetQ/DmetEF strains in their ability to colonise the mouse nasopharnyx. In a mouse model of systemic infection although significant infection was established in all mice, there were reduced spleen bacterial CFU after infection with the DmetQ/DmetEF strain compared to the wild-type strain. These data demonstrate that Sp_0149 encodes a high affinity methionine ABC transporter lipoprotein and that Sp_0585 – Sp_0586 are likely to be required for methionine synthesis. Although Sp_0149 and Sp_0585-Sp_0586 make a contribution towards full virulence, neither was essential for S. pneumoniae survival during infection., This work was undertaken at UCLH/UCL, which received a proportion of funding from the Department of Health's NIHR Biomedical Research Centre's funding scheme, and was supported by the UCL Charities funding and grants from the British Lung Foundation (P05/3) and the Wellcome Trust (grant reference 076442).