- Volume 2, Issue 7A, 2020
Volume 2, Issue 7A, 2020
- Abstracts from Annual Conference 2020
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- Oral Abstract
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Strategies for glycan acquisition by Bacteroidetes in the soil: The carbohydrate enzymology of Chitinophaga pinensis
More LessThe secretion of extracellular enzymes by soil microbes is rate-limiting in the global recycling of biomass. Fungi and bacteria compete and collaborate for nutrients in the soil, with wide ranging ecological impacts. Within soil microbiota, the Bacteroidetes tend to be a dominant bacterial phylum, just like in human and animal intestines. The enzymology of Bacteroidetes in the dynamic and competitive soil environment is under-explored compared to their cousins from the human and ruminant gut ecosystems. We are exploring carbohydrate binding and deconstruction by Chitinophaga pinensis. This species was isolated from the leaf litter of a pine forest, and our ongoing microbiological, biochemical, and proteomic analyses show that C. pinensis has a marked metabolic preference for carbohydrates (glycans) of microbial, rather than plant, origin. The species has a repertoire of enzymes that degrade components of the fungal cell wall, and we are characterising several important enzyme activities, including some with unusual substrate specificity.
Several features of the C. pinensis “cazome” make it note-worthy. In particular, there is a significantly reduced reliance on the Polysaccharide Utilisation Loci that define glycan acquisition in most well-studied gut symbiont Bacteroidetes. Instead, C. pinensis produces some large multi-modular enzymes that convey multiple complementary carbohydrate-binding and -degrading functions, and which are often secreted via the phylum-specific Type IX Secretion System.
This presentation will highlight our latest enzyme characterisation data, discussed in the context of the environmental functions of soil bacteria, as well as the use of enzymes for industrial biotechnology.
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Phenotypic characterization and ecological succession of microorganisms during the fermentation of Cassava and Maize
More LessFermented foods are consumed by a very large population in Africa but the products have many drawbacks ranging from shelf life instability to contamination and toxicity. These foods therefore require an upgrade through improved fermentation processes. This work determined the phenotypic characteristics of the fermenting microorganisms and microbial ecological succession during fermentation of cassava and maize to determine the predominant fermenting microorganisms. Cassava roots and maize grains were fermented using the traditional method of processing them into fufu and ogi for 72 h and 48 h respectively. Samples were drawn every 12 h for analysis. Enumeration and characterization of lactic acid bacteria were carried out on MRS medium with subsequent microscopic examination, physiological, biochemical reaction tests and API 50 CH gallery. Yeast isolates were identified by their morphological characteristics. Thirteen lactic acid bacteria were isolated from the fermenting cassava and 6 from the fermenting maize. The Isolates were Gram positive and catalase negative. Lactobacillus plantarum, L. fermentum and L. pentusus predominated in both fermentations while Candida tropicalis, C. krusei and Saccharomyces cerevisae also predominated in both fermentations. Candida inconspicuo was found only in cassava fermentation. The results of this work revealed the microbial ecology of fermented cassava and maize which is a prerequisite to the understanding needed to develop a multifunctional starter culture for these fermentations for their upgrade.
Keywords: Cassava, Maize, Fermentation, lactic acid bacteria, Yeasts.
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Galleria mellonella – a novel infection model for the study of Neisseria gonorrhoeae virulence and pathogenicity”
More LessGonorrhea is the second most commonly reported notifiable sexually transmitted disease in the world. Neisseria gonorrhoeae causes 78 million cases annually of gonorrhoea worldwide. There are no vaccines and antibiotic-resistant organisms are circulating rapidly. The estradiol-treated female mouse model is the only animal model available for studying the host response against gonococci and biological significance of host-restricted bacterial-host cell interactions observed in vitro. However, mouse models have limitations such as cost, time and ethics. Therefore, there is an urgent need for the development of new alternative in vivo models. The Galleria mellonella larval model is a simple, widely available, cost-effective, and powerful tool for studying microbial infections prior to any vertebrate animal testing. Here we report, for the first time, that G. mellonella can be used as an infection model for Neisseria spp., focusing particularly on N. gonorrhoeae. We demonstrated dose-dependent larval death and recovery of viable gonococci from the host, visualised host-pathogen interactions using histopathology and confirmed the importance of insect haemocytes as an innate immune cell during infection. The model was also used to test the efficacy of antibiotics used to treat gonorrhoea. Our results demonstrate that G. mellonella can be used as a model to study pathogenesis and virulence of gonococcal infection in addition to rapid in vivo testing of antimicrobials.
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In vitro reassortment and adaptation of influenza A viruses circulating in swine
Since the last influenza pandemic in 2009, H1N1pdm has been introduced into the swine population in Europe where, in combination with swine influenza A virus (IAV) lineages, it started to generate a variety of reassortant viruses of unknown zoonotic risk for humans. To study these reassortment events, we isolated a wild swine lung cell clone (C22) susceptible to IAV infection. We established conditions for co-infection and passaging of H1N1pdm and swine avian-like H1N1. After 7 passages, we plaque-purified C22-adapted strains, characterized their genome composition by next-generation sequencing and analysed replication abilities in swine and human lung cell lines as well as in human lung tissue ex vivo.
Among C22-adapted viruses isolated from co-infection, we revealed reassortants carrying PB1/PA/NA or only PB1/PA from H1N1pdm. We also detected exclusively swine H1N1-derived strains. All isolates carried distinct mutations. As expected, adapted viruses reached higher titers compared to both parental strains in swine lung cells. Furthermore, all C22-adapted viruses were able to replicate in human lung A549 cells without any prior adaptation to the human host. Strikingly, all reassortants were able to infect and efficiently replicate in human lung tissue ex vivo, indicating that these viruses might pose a zoonotic risk.
To summarize, we successfully established an in vitro swine-like model to study reassortment and adaptation of IAVs currently circulating in swine. Our results indicate that our model might be a useful tool to prospectively evaluate the compatibility of different IAV strains to generate reassortants, which might represent a threat to the human population.
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Assessment of Recycling-derived Fertilizers as an Alternative to Mineral Fertilizers – Effects on the Soil Microbiome
More LessPhosphorus (P) is an essential macronutrient for all living organisms and is applied as fertilizer in agroecosystems to improve crop growth. Recycling-derived fertilizers (RDFs) have been developed for nutrient recovery from Europe’s largest waste streams as a sustainable alternative to this finite resource. The impact of four RDFs (two ashes, two struvites) on the soil microbiome in comparison with a P-free control and triple super phosphate (TSP) as mineral fertilizer was investigated in a pot trial and a subsequent microcosm trial (subset of samples). For both experiments perennial ryegrass was cultivated for 54 days. The pot trial was conducted at P fertilization rates of 20 and 60 kg P ha-1 in quadruplicates. After the pot harvest the bulk soil was stored until the microcosm trial was conducted, using the control, TSP and the two ashes at 60 kg P ha-1 in six replicates. Pot trial results showed highest P bioavailability from struvites at high P rates, also resulting in higher biomass yield on average. Furthermore, P solubilization capabilities from tri-calcium phosphate was enhanced in the RDFs treatments, while the TSP treatments were negatively affected. For the microcosm trial, most probable number (MPN) analysis showed that phytate-utilizing bacterial abundance was significantly increased in one of the ashes and had also remained higher in the RDF treatments after storage. Understanding the effects of recycling-derived fertilizer application on the soil P cycle is vital for developing a more sustainable agriculture.
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Investigating the mechanisms of BK polyomavirus egress and virus-host interactions
More LessBK polyomavirus (BKPyV) is a small, non-enveloped dsDNA virus that infects 70-90% of the world's population and causes a lifelong, silently persistent infection. In immunocompromised individuals, BKPyV replication can result in serious pathology. Bone marrow transplant patients can develop a haemorrhagic cystitis, and in kidney transplant patients BKPyV replication can provoke a nephropathy that leads to deterioration of allograft function and eventual loss of the transplanted organ. There are currently no antiviral treatments with clinical efficacy against BKPyV associated nephropathy.
While the life cycles of non-enveloped viruses are often assumed to require cell lysis to release progeny virions, we have evidence to suggest that BKPyV exits the cell via non-lytic means using an unconventional secretory pathway. We have investigated the effects of knocking out cellular genes thought to be involved in unconventional secretory pathways that bypass the Golgi apparatus on the release of BKPyV. We observe decreased BKPyV release from cells that have undergone CRISPR-mediated knockout of Golgi Reassembly Stacking Protein (GORASP) 1 or 2. Investigation of BKPyV-induced changes to the plasma membrane of infected cells demonstrated increased cell surface expression of transmembrane proteins normally resident in the endoplasmic reticulum. This appears to be inhibited by the knockout of GORASP1 or 2, suggesting that virions and ER markers are secreted via a common pathway in infected cells. These experiments are uncovering novel virus-host interactions that, when targeted, could help prevent BKPyV-associated nephropathy and allograft loss.
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- Poster Presentation
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How to build a virulence factor: the role of three novel enzymes in the biosynthesis of the Group A Carbohydrate
More LessThe Group A carbohydrate (GAC), a bacterial surface polysaccharide, is an essential virulence factor of Streptococcus pyogenes required for growth and infection of humans.In terms of its chemical composition, this peptidoglycan-anchored polymer is mainly formed by a string of rhamnose sugars, with alternated modifications of N-acetylglucosamine and glycerolphosphate. The rhamnose polysaccharide (RhaPS) that forms the backbone chain is synthesised intracellularly by the sequential action of three rhamnosyltransferases named GacB, GacC and GacG. Importantly, deletion of any of these rhamnosyltransferases causes bacterialdeath.
In this work, we used an interdisciplinary approach to demonstrate that: 1) GacB is a novel enzyme that initiates the RhaPS biosynthesis; 2) GacC catalyses the formation of a unique stem; 3) GacG elongates the RhaPS string by adding a yet unknown number of rhamnoses. Here, we also show that homologs from different streptococcal species can substitute GacB and GacC in the RhaPS production. In particular, we demonstrate that several human pathogens from the Streptococcus genus encompassed in the Lancefield serotyping scheme, and the dental pathogen Streptococcus mutans can replace S. pyogenes’ enzymes. In contrast, the homologs from S. pneumoniae sp. D39 did not, suggesting a different structural arrangement for its surface carbohydrate. Our results highlight the importance of the group carbohydrate biosynthesis pathways in the Streptococcus genus and open the door for the future development of multi-target compounds that could inhibit these enzymes in Streptococcus pyogenes and other pathogenic streptococci of clinical and veterinary importance.
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Effects of ultraviolet-C on the spores of Bacillus subtilis and Bacillus velezensis suspension in phosphate buffered saline with their structural and molecular analysis using Raman-AFM imaging
More LessBacterial spores are of concern in food processing due to their ubiquity and resistance. This study seeks to determine the effect of ultraviolet C (UV-C) in the inactivation of spores of Bacillus subtilis and Bacillus velezensis that can result in enzymatic spoilage in foods using PBS as the suspension medium. Purified spore samples were treated under 1 pass in a UV-C reactor using 10 mL of spore inoculum with one dose of the radiation (410 mJ/cm2) for 10secs at room temperature. Aliquots of the treated samples were plated on tryptone soy agar supplemented with 0.6% glucose and the colonies counted. Flow cytometry analysis was done using 500 μL of both treated and control samples with a cell concentration of a ≥106 CFU/ml with propidium iodide (15 μM) and SYTO 9 (500 nM) used as live/dead stains. Samples were processed for microscopy (SEM and Raman-AFM Imaging). The maximum lethality is 2.5 for B. velezensis and the minimum is 0.1 for B. subtilis. Microscopic imaging of treated spores shows significant morphological disruption of the spore structure. The Raman spectroscopy analysis reveals the B. subtilis isolates to have the highest concentrations of dipicolnic acid (Ca+2DPA) as well as other compounds belonging to other functional groups. Flow cytometric analysis of treated spores reveals sub-populations unaccounted for by plate count. UV-C shows a promising application in the inactivation of resistant spores during processing of liquid foods such as milk.
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The use of wound dressings as a means to alter the composition of biofilms in a chronic wound model
More LessIt is estimated that £5 billion are invested yearly into chronic wound management by the NHS. Whilst the demand for treatment rises every year, it has become harder to treat wounds given the burden of antimicrobial resistance. Chronic wounds can easily become harbouring grounds for polymicrobial biofilms in which species interact in specific ways.
This study assessed the interactions between two commonly co-isolated chronic wound pathogens: Pseudomonas aeruginosa (ATCC 9027) and Staphylococcus aureus (EMRSA 15), whose biofilm relation initiates a Gram-negative shift. During this phenomenon, P. aeruginosa takes over the majority of the bacterial community, at the detriment of S. aureus. The Gram-negative shift marks the turning point from an acute to a chronic wound. The pH of a chronic wound is typically alkaline, and it was hypothesised that topical dressings with an acidic pH could disrupt the onset of the Gram negative shift, and therefore chronicity. Six different topical dressings with low pH were used in achronic wound model to assess their ability to reverse or delay the Gram-negative shift. It was found that they did not have an impact on the onset of the Gram-negative shift, despite their low pH values. However, the lower the pH of the dressings, the more frequently small colony variant (SCV) bacteria were observed in the biofilm. SCVs are known for causing persistent or chronic infections. It was therefore concluded that low pH dressings alone may not be favourable for managing chronic wound infection.
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In silico prediction and in vitro assessment of microbial substrate utilisation: a focus on newly identified health promoting gut bacteria
More LessThe contribution of the gut microbiota to health and disease is becoming ever more apparent in the last number of years, due to developments in DNA sequencing technology and more well-defined cultivation techniques. This has resulted in the identification of health-promoting bacteria. Until recently, prebiotics, non-digestible food substrates which are selectively utilised by beneficial bacteria, were employed with a view to increasing the growth of well-established health promoting bacteria, namely Lactobacillus and Bifidobacterium. However, other beneficial bacteria recently revealed may also be targeted to enhance their growth as they establish themselves as the next generation of health-promoting microbes. These include anaerobes such as Akkermansia muciniphila, Faecalibacterium prausnitzii and Eubacterium rectale. Identification of growth substrates/bioactives through the analysis of genome sequence data can aid in elucidating which substrates may best enhance the growth of these microbes which are often difficult to grow.
The phenotypic microbial trait analyser, Traitar, can predict 67 phenotypes based on the genome sequence inputted. Some of these traits include substrates that could potentially be utilised by the bacteria. Another tool, CarveMe, which has been created with the aim of making metabolic modelling more user-friendly, was also used with the same genomes. A select number of substrates identified in both tools have been chosen to be evaluated in vitro in order to establish the accuracy of these predictive tools as well as giving an indication as to how these beneficial microbes can be modulated through dietary components.
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Effects of Vibrio cholerae infection and colonization on the zebrafish intestinal microbiome
More LessZebrafish (Danio rerio) are an attractive model organism for a variety of scientific studies, including host-microbe interactions. Zebrafish contain a core (i.e., consistently detected) intestinal microbiome consisting primarily of Proteobacteria. Furthermore, this core intestinal microbiome is plastic, and can be significantly altered to due external factors. The organism is particularly useful for the study of aquatic microbes that can colonize vertebrate hosts, including Vibrio cholerae. As an intestinal pathogen, V. cholerae needs to colonize the intestine of an exposed host for any type of pathogenicity to occur. It is suspected that members of the resident intestinal microbial community need to be eliminated by V. cholerae in order for colonization, and subsequently disease, to occur. While numerous studies have explored various aspects of the pathogenic effects of V. cholerae on zebrafish and other model organisms, few, if any, have examined how a V. cholerae infection alters the resident intestinal community. In this study, 16S rRNA gene sequencing was utilized to investigate how various strains of V. cholerae alter the aforementioned microbial profiles following an infection. We found that V. cholerae infection and subsequent colonization induced significant changes in the zebrafish intestinal microbiome, with specific members of the microbial community targeted. Additional salient differences to the microbial profile were observed based on the particular strain of V. cholerae utilized for challenging the zebrafish hosts. We conclude that V. cholerae causes significant modulation to the zebrafish intestinal microbiome in order for infection and subsequent disease to occur.
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Aeromonas caviae motility and glycosylation
More LessAeromonas are Gram-negative facultative anaerobic rods, which inhabit various aquatic environments and are pathogens of both warm and cold-blooded animals. In humans they cause gastro-enteritis and wound infections. They are motile in liquid environments by a single polar type of flagellum. The flagellum plays an important role for the bacterial colonisation and the adhesion to the host cells. The Aeromonaspolar flagella filament is a polymer composed of two flagellins, FlaA and FlaB. The flagellins are O-linked glycosylated through the addition of the unusual bacterial sugar pseudaminic acid to serine and threonine residues within the flagellins D2/D3 domain. The addition of this sugar is essential for flagella filament assembly and bacterial motility. The flagellin’s are modified by between 6 – 8 sugar residues that occupy the potential 14 sites of attachment. Motility accessory factors (Maf proteins) are candidate enzymes for transferring glycan molecules to the flagellin (glycosyltransferases transferring sugar to flagellin) due to their genetic location and motility phenotypes associated with disruption mutants.
This study utilised site-directed mutagenesis to change the potential sites of flagellin glycosylation to assess the effect of these mutations on motility by swimming assays and flagella filament formation by electron microscopy. The analysis of different numbers of site-directed mutants suggest that some sites are more important than others and that the removal of 4 sites results in greatly reduced motility.
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Antimicrobial resistance Salmonella isolates recovered from food products of animal origin in the Russian Federation
More LessThe study was aimed at Salmonella isolation from samples of animal food products submitted for testing from various regions of the Central part of the RF and serotyping of the recovered isolates and their testing for antibiotic resistance. A total of 2,342 tests were performed and 87 (3.7%) Salmonella isolates were recovered. Most of them (54 isolates) were recovered from poultry meat and poultry meat preparation samples submitted for testing. Besides, 25 isolates were recovered from pork and pork preparation samples, 7 isolates – from beef samples, 1 isolate – from hard cheese samples. Serotyping of 64 Salmonella isolates showed that the majority of the isolates (57.8%) belonged to О7 group. Also, Salmonella isolates belonging to О9 (21.9%), О8 (9.4%), О4,5 (6.2%) and О10 (4.7%) were detected in food products. S. Enteritidis, (23.3%), and S. infantis (18.7%), were predominant based on the number of detections. Also, the following serovars were identified: S. typhimurium, S. nigeria, S. montevideo, S. derby, S. meleagridis, S. virchov, S. oranienburg. Tests of 87 Salmonella isolates for their antibiotic resistance with disk diffusion method revealed that they were highly resistant to nalidixic acid (70.1%), tetracycline (49.4%), trimethoprim/sulfamethoxazol (40.2%). Moreover, nalidixic acid-resistance was common for all identified isolates. Seventeen isolates (19.5%) demonstrated multiple antibiotic resistance and two isolates were found to be resistant to ≥7 antibiotics. All recovered isolates were susceptible to gentamicin, amikacin, meropenem and imipenem. Obtained results indicate the necessity of Salmonella antibiotic resistance monitoring to gain understanding of Salmonellas’ antibiotic resistance emergence and trends.
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Bacterial profile of meconium of neonates born at National Referral Hospital Cipto Mangunkusumo, Jakarta, Indonesia
Microbial colonization of a neonate’s gastrointestinal tract has significant perinatal and lifetime health consequences, with some clinical outcomes that have been linked to differences in the diversity and composition of gut microbiota. The effort to engineer intestinal ecosystem has led us to preserve the cultivable commensal microbiota. Here we investigated the association of cultivable bacterial diversity of neonates meconium from Indonesian National Referral Hospital Cipto Mangunkusumo (NRHCM) with mode of delivery and feeding patterns, as well as hyperbilirubinemia. We performed a cross-sectional study of meconium and clinical data collected from 14 Indonesian neonates born at NRHCM. Culture-dependent identification of bacterial isolates was conducted by performing simultaneous microbiological and molecular 16S rDNA PCR-Sanger sequencing methods. Phylogenetic tree and principal components analysis were employed to determine the bacterial profile and their association with clinical characteristics and outcomes. Cultivable bacterial profile indicates the predominance of Firmicutes (84,41%), with an abundant population of Staphylococcus (53.24%) with top three most significant population present are, i.e. S. hominis (12.99%), S. epidermidis (11.68%), and S. haemolyticus (10.39%). Bacterial diversity was associated with mode of delivery which showed that vaginal route populated by lower diversity of cultivable bacteria but by fewer opportunistics one than that of cesarean, with Staphylococcus hominis dominates the population, whereas with feeding patterns showed that the exclusive breast-fed was most populated by Staphylococcus, whereas non-exclusive one shared the same proportion of Staphylococcus and Bacterioides. While, non-hyperbilirubinemia group showed more abundant and diverse Staphylococcus than that of the opposite group.
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A family of T6SS antibacterial effectors related to L,D-transpeptidases targets the Peptidoglycan
Type VI secretion systems (T6SSs) are contractile nanomachines widely used by bacteria to intoxicate competitors. Salmonella Typhimurium encodes a T6SS within the Salmonella pathogenicity island 6 (SPI-6) that is used during competition against species of the gut microbiota. We characterized a new SPI-6 T6SS antibacterial effector named Tlde1 (type VI L,D-transpeptidase effector 1). Tlde1 is toxic in target-cell periplasm and its toxicity is neutralized by co-expression with immunity protein Tldi1 (type VI L,D-transpeptidase immunity 1). Time-lapse microscopy revealed that intoxicated cells display altered cell division and lose cell envelope integrity. Bioinformatics analysis showed that Tlde1 is evolutionarily related to L,D-transpeptidases. Point mutations on conserved histidine121 and cysteine131 residues eliminated toxicity. Co-incubation of purified recombinant Tlde1 and peptidoglycan tetrapeptides showed that Tlde1 displays both L,D-carboxypeptidase activity by cleaving GM-tetrapeptides between meso-diaminopimelic acid3 and D-alanine4, and L,D-transpeptidase exchange activity by replacing D-alanine4 for a non-canonical D-amino acid. Tlde1 constitutes a new family of T6SS effectors widespread in Proteobacteria. This work increases our knowledge about the bacterial effectors used in interbacterial competitions and provides molecular insight into a new mechanism of bacterial antagonism.
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Assessment of mosquito insecticidal activity of bacterial extracts produced by Colombian strains
Aedes aegypti and Aedes albopictus are the major vectors for the transmission of more than 30 disease-causing viruses as dengue fever, dengue hemorragic fever, zika, yellow fever and chikungunya. Vector control is one of the important strategies used in order to fight these diseases in tropical and subtropical countries. However, mosquito control is facing a threat because of the emergence of resistance to synthetic insecticides. The aim of this study was to identify larvicidal and adulticidal activity of secondary metabolites in extracts produced by bacteria isolated from different sources in Colombia. A total of 105 extracts produced from the same number of bacteria were evaluated for their activity against fourth instar larvae and adults of A. aegypti and A. albopictus using standard protocols defined for the WHO (World Health Organization) and CDC (Control Disease Centre, USA). Six extracts showed relevant activity (more than 50% of mosquito larvae were killed after 48 hours), two of them showed to be actives against larvae of Aedes aegypti and four against larvae of Aedes albopictus. None of the extracts showed activity against the mosquitoes in adult stage. The bacteria producing active extracts were identified using the biolog ® identification system as Serratia marcescens ss marcescens, Escherichia hermannii, Serratia marcescens ss marcescens, Bacillus marisflavi, Bacillus atropheus/subtilis and Pseudomonas chlororaphis subsp. aurantiaca. In conclusion, bacterial extracts are a good source for the search of new strategies in the control of mosquitoes. Further studies to determine the compound responsible for the insecticidal activity are in progress.
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Screening for urinary tract infection-causing Klebsiella pneumoniae using CHROMagar
More LessIntroduction. Urinary tract infections (UTIs) are considered prevalent among humans. While Klebsiella pneumoniae and Escherichia coli are commonly isolated from patients with UTIs, K. pneumoniae is more frequently isolated from specimens isolated from hospital-acquired infections.
Aim. The current study aimed to characterise and evaluate pathogenic K. pneumoniae isolated from patients suspected with a UTI at King Abdulaziz University Hospital.
Methodology. Twenty-four urine samples obtained from patients, between 12 November 2018 and 11 January 2019, were included in the study, and the microbial content was analysed via urine culture and VITEK 2 analyses.
Results. Of the 24 urine specimens, 23 samples (95.8%) yielded significant microbial growth (> 105 CFU/mL K. pneumoniae), and one sample (4.2%) yielded non-pathogenic microbial growth (< 105 CFU/mL K. pneumoniae). Of the specimens with cell counts of >105 CFU/mL, 21 samples (87.5%) were pure cultures showing the growth of a single pathogen, and three samples (12.5%) were mixed cultures, showing the growth of two or more pathogens; 10 (41.7%) samples were extended-spectrum beta-lactamase (ESBL) producers. VITEK 2 analysis showed that the most effective antibiotic was piperacillin, with 87.5% of the strains isolated in this study showing sensitivity to it, followed by gentamicin (83.3%) and ciprofloxacin (79.2%). Antibiotic susceptibility studies identified resistance against ampicillin (45.83%), trimethoprim (41.67%), and amoxicillin (25%) in the study isolates. Differential chromogenic culture media CHROMagar ESBL and CHROMagar KPC (K. pneumoniae carbapenemase) were used for rapid screening of ESBL and KPC producers. Among the 24 K. pneumoniae isolates, six isolates (25%) formed metallic blue colonies on CHROMagar ESBL, five (20.3%) were inhibited, and nine (37.5%) showed weak pigmentation. Three isolates (12.5%) formed pink colonies on CHROMagar ESBL. Seventeen isolates (70.8%) were KPC-positive, four (16.7%) showed weak pigmentation, and three (12.5%) formed pink colonies on CHROMagar KPC. The results from CHROMagar cultures agreed with those of VITEK 2 analyses.
Conclusion. Various microbial detection methods have been used in medical microbiology laboratories to identify and screen for microbial resistance in clinical specimens. VITEK 2 and CHROMagar are amongst the commonly used methods. Here, we report a possible discrepancy between the detection techniques and that the data obtained by different methods need not be synergistic. However, the use of a chromogenic medium offers a quick and accurate method for detection, enumeration, and presumptive identification of urinary tract pathogens.
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Skin microbiome profiling and cultivation of healthy adult Indonesian skin
More LessStudies on the impact of skin microbiota on human health have been gaining more attention. The skin microbiome is considered to provide several probiotics for skin therapeutic. Beneficial bacteria mixtures (bacterial cocktail) isolated from targeted organs have shown promising modulatory activities for use in skin therapeutics. The objectives of this study were to determine and identify the microbial communities on the skin that can be potentially used as probiotics-postbiotics. Determination and identification of skin microbiota were carried out simultaneously by employing next-generation sequencing (NGS) of direct sampling, as well as by bacterial cultivation; twenty bacterial isolates with different characteristics were selected and identified by both culture-based methods and 16sRNA sequencing. We found that Actinobacteria and Firmicutes are the most abundant phylum present on the skin as presented by NGS data, which constitute to 67% and 28.59% of the whole bacterial population, consecutively. Three strains, i.e., Staphylococcus hominis (AN MK968325.1), Staphylococcus warneri (AN MK968315.1), and Micrococcus luteus (MK968318.1), were obtained from cultivable samples. They were potential to be developed further as probiotics in skin microbiome therapeutic as well as for postbiotic formulation. However, the promising formula of bacterial cocktail for skin microbiome therapeutic must be elucidated thoroughly to avoid unwanted effects by performing visual observation on agar plate and by molecular approach, q-PCR.
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Predominance of Streptococcus pneumoniae among TB patients complicates Dots chemotherapy at infectious diseases hospital Kano, Northwest Nigeria
More LessDirectly observed treatment Short course regimen has been know to be efficacious in the chemotherapy management of tuberculosis. However, co- infection with other opportunistic bacteria could limit this success to the level of clinical suspecting the emergence of false positive multidrugs resistant TB among the patients which in many cases can lead to establishment of erroneous treatment schedule. Accordingly this facility based surveillance monitors co-infection with other bacteria in TB patients attending Dots clinic at IDH Kano, Nigeria with view to ascertaining the drug susceptibility so as to proffer better chemotherapy strategies. A total 35 non - duplicated positive AFB sputum sample collected from patients with pulmonary tuberculosis on Dots where processed according to standard bacteriological procedures including macroscopic, culture and microscopic examination using both AFB gram staining and culture methods for screening and isolation of other bacterial pathogens. These were confirmed biochemically. Overall 54.3% of the patients were Still AFB positive, Streptococcus pneumoniae and Staphylococcus species we’re predominat in up to 84.2% and 15.8% of the patients respectively. This study indicated secondary bacterial pathogenesis with pulmonary symptoms resembling TB among the patients including those that were even isolated for being refrectory to Dots an false positive conclusion of drug resistant TB. However, ofloxacin and ceftriaxone which show appreciable potencies against secondary bacterial pathogens identified where used to treat successfully, patients claimed posses drug resistant TB. It’s imperative that, success of Dots therapy could be improved by augumentin with very potent non TB antibacterial such as Streptococcus pneumoniae regimen.
Keys. TB.
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Comprehensive evaluation of the antigenic impact of intra-genotypic human papillomavirus variant diversity on recognition by neutralizing monoclonal antibodies raised against lineage a L1 virus like particles
Introduction. Naturally-occurring variants of Human papillomavirus (HPV) genotypes have been defined as lineages and sub-lineages, but little is known about the impact of this diversity on protein function. We have previously demonstrated that variation within the major (L1) and minor (L2) capsid proteins impact the susceptibility of HPV to serum antibodies elicited by vaccination and natural infection. Higher resolution mapping of variant residues, however, requires the availability of appropriate tools, such as type-specific monoclonal antibodies (MAbs). These empirical data will improve our understanding of the consequences of natural variation on capsid antigenicity.
Methods. We investigated the susceptibility of 37 representative pseudovirus variants of HPV16, HPV18, HPV31, HPV33, HPV45, HPV52 and HPV58 to neutralization by type-specific murine MAbs raised against the A lineage of their respective genotypes. Homology models derived from available HPV L1 crystal structures were generated to permit mapping of variant residues onto the surface-exposed L1 protein for relevant variants
Results. Type-specific lineage A-specific MAbs demonstrated differential reactivity against some, but not all, variants within its respective genotype. Some of these differences were minor (<4 fold) while some variants displayed orders of magnitude reduced sensitivity. These differences in antigenicity were mapped to a limited number of variant residues on the capsid surface.
Conclusions. These data contribute to our understanding of HPV L1 variant antigenicity and may have implications for seroprevalence or vaccine immunity studies based upon L1 antigens.
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Molecular genetics of the virulence plasmids of pathogenic Escherichia coli O104:H4
More LessIn 2011 a large outbreak of enterohemorrhagic gastroenteritis and haemolytic uremic syndrome (HUS) throughout Europe resulted in almost 4,000 infections, 845 cases of HUS and 54 fatalities. This was due to a dangerous exchange of mobile genetic elements (MGE) resulting in a hybrid strain of E. coli O104:H4. This strain carried an unusual combination of EAEC- and STEC-associated virulence factors on a plasmid and phage respectively. In vitro the virulence plasmid has exhibited unusual stability under a wide range of environmental stresses, contrasting with rapid plasmid loss in the human gut. This project will characterise plasmid encoded maintenance systems responsible for its unique stability. Current investigations focus on toxin-antitoxin (TA) systems involved in postsegregationalkilling which contribute to plasmid maintenance therefore resulting in increased virulence. By inducing expression of putative TA genes cloned onto lab-made plasmid vectors, we have analysed their function in the cell. Once characterised we will further investigate the effects of various environmental conditions able to disrupt these TA systems, ultimately resulting in plasmid loss. This atypical strain displayed heightened pathogenicity and providedun foreseen treatment challenges. We aim to further our understanding of MGE carriage in O104:H4 as a model to predict and combat future outbreaks of hybrid pathovars.
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The Type VI secretion system in commensal Neisseria spp.
More LessA type 6 secretion system (T6SS) was recently identified in the genome sequence data of an isolate sourced from a throat swab of a volunteer that is believed to be N. subflava. The T6SS is one of the most recently discovered bacterial secretion systems and this is the first time it has been reported in Neisseriaceae. Since this discovery, genome sequence analyses for a number of other commensal Neisseria spp. has identified that in fact, two distinct T6SS types exist across Neisseriaceae. These two types are clearly defined and are different to one another in both their core gene sequences and organisation. The two systems also differ in the number of VgrG proteins required for toxic effector protein delivery, as well as type of effector associated with them. The predicted VgrG/effector combinations identified in our isolate are not common to all members of the same species and further analysis has identified a wide range of diversity in these components between different strains of the same species. The data provides possible evidence that T6SS positive commensal Neisseria spp. can acquire new VgrG/effector combinations through competitor killing. A number of putative effectors have so far been identified within the genome of our original isolate, including hydrolases, phospholipases, and nucleases. Whilst these are predicted to be antibacterial effectors, the conditions under which the T6SS system is activated, as well as demonstration of the function of the effectors still needs to be investigated experimentally.
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Failing to control Maedi-Visna
Maedi-Visna is a lentivirus of sheep that causes lung disease and chronic wasting. It has been designated an “Iceberg disease” by the UK sheep industry levy board with a very large burden of subclinical disease that is often not apparent until losses in an individual flock become catastrophic. Disease prevalence in the UK is thought to have doubled in the last 10 years, however farmer and veterinary awareness of the disease is poor. There is no vaccine and treatment is not cost effective, meaning that the only realistic control option is culling of affected animals.
Current testing protocols use MV gag protein ELISAs. A long lag time between infection and antibody production means that many animals are missed on flock screening and repeated rounds of testing over a period of years are necessary remove all infected animals. Preliminary testing of flocks that have attempted eradication indicates that those that do not keep testing until all animals are negative fail to eliminate the disease and that prevalence rates can even increase substantially in these flocks. The viruses extreme variability confounded attempts to develop a qPCR capable of detecting all variants, indeed deep sequencing was required to establish which strains of virus are currently present in UK sheep as there has been substantial genetic drift since the last sequencing studies (performed more than 20 years ago). More promisingly virus was detectable in nasal swabs of experimental animals at least offering a possibility for sampling methods that can be done by farmers themselves.
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Testing for novel inhibitors of periodontitis-associated sialidases
More LessThe microorganisms associated with severe periodontitis are the periodontal pathogens of the red complex: Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola. These organisms cleave sialic acids found at the terminal end of host glycoconjugates by hydrolysing the glycosidic linkages with their expressed sialidases, thereby affecting the integrity of the host periodontium and promoting disease progression. Both P. gingivalis (SiaPG) and T. forsythia (NanH) sialidase enzymes were purified using HisTag affinity chromatography and a range of putative synthetic and plant-based inhibitors were tested for their ability to inhibit both enzymes using a MUNANA cleavage assay. Investigation of sialidase inhibitory activity of these compounds revealed that the plant derived alkaloids: Epicatechin gallate (IC50 = 21.75μM and 120.9μM) and Berberine chloride (IC50 = 106.2μM and 125.5μM) were more effective inhibitors of both SiaPg and NanH enzymes than the anti-influenza drug Zanamivir, an FDA approved viral neuraminidase inhibitor. Finally, a range of newly synthesized sialic acid analogues were effective in the micromolar to nanomolar range against both SiaPg and NanH enzymes with compound 2e3aDFNeu5Ac9N3 having an IC50 of (3.846μM and 49.40nM) respectively. The data suggests several novel inhibitors of these enzymes that might have future use as novel drugs against diseases such as periodontitis, and which we are currently testing further in host-pathogen interaction studies.
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Coccus Pocus 2019: A microbiology-inspired scary story competition
More LessAntibiotic resistance by pathogenic microorganisms is a major threat of our times, leading to a significant rise of serious untreatable infections, especially in hospital environments. In addition, biofilms further protect the microbes against antibiotics, detergents and the attacks of our immune system. This Halloween, we launched an exciting scary story competition, named Coccus Pocus 2019. The participants were encouraged to write a short horror sci-fi story between 500 and 2,000 words, including antimicrobial resistance and microbial biofilms. The evaluation committee that was composed of eight academics and researchers from the University of Hull and other institutions, ranked the stories according to intrigue of their plot, use of language, character description and scientific soundness. The prizes (online gift vouchers) were awarded to the three winning stories, during an awards ceremony. Feedback questionnaires were completed by the participants, which showed that they all found the competition very interesting and useful, allowing them to sharpen their creative writing skills and explore key microbiology topics. The event was communicated in the social media and blogs were posted in university bulletins and on microbiology websites. It is our ambition that the competition will be held again and again around the country, aiming to increase public awareness about the important problem of antimicrobial resistance and biofilms and boost the enthusiasm of young people about the fascinating field of microbiology.
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Intelligent wound dressing for diagnostic & therapeutic applications - critical to wound infection
More LessA medical device comprising of biomaterials responsive to biochemical stimuli: channel for indicating the infective states of wounds and ensuring delivery of smart antimicrobial and antibiofilm agents to promote tissue regeneration and healing.
The importance of providing diagnostic wound dressings that can inform healthcare professionals on the state of infection within wounds but also provide some of the treatment required in response to at risk or infected wounds is of key interest. The aim is to investigate an innovative proof of concept diagnostic and detection system, an intelligent hydrogel wound dressing that responds to specific biochemical stimuli in wounds (MMPs and pH) enabling the selective and triggered release of antibiofilm and antimicrobial agents (‘Detect and Treat’)to the trauma site. The dressing is made of a sterile alginate core material covered in a biocompatible dry or hydrated peptide-polymer-complex film and may include a fluorescent dye which upon release during the wound healing process indicates when a change in dressing is necessary. Efficacy studies of the hydrogel dressing were performed within a drip-flow bioreactor in which regression of Pseudomonas aeruginosa biofilm was observed. A 5-log reduction in biofilm was observed in comparison to an untreated control biofilm. The hydrogel dressing indicated a clear response when in contact with biofilms produced only by pathogenic strains of bacteria when analysed. This further confirmed the adequate release and function of the antimicrobial and antibiofilm agents within the peptide-polymer-complex formulation of the hydrogel wound dressing.
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Epidemiological studies of malaria parasite on HIV patients attending General Hospital Awo-Omamma, Oru East, Imo State,Nigeria
More LessHIV and malaria are the two most prevalent and deadly diseases in the world. Malaria and HIV accounted for about 255 million cases in 2017, with malaria having 86% of this distribution and HIV having 14% of the distribution. Given the overlap of their geographic distribution and resultant rates of coinfection, interactions between the two diseases pose major public health problems. This study was aimed at investigating the epidemiology of malaria –HIV co-infection in respect to sex, age and its association with CD4+ count and viral load. 230 HIV sero-positive participants and 100 HIV sero-negative participants(control) were employed for this study. 52 (22.6%) of the HIV infected participants tested positive for malaria while only 9(9.0%) of the non-HIV participants tested positive to malaria. The prevalence of malarial infection in HIV positive individuals was higher in females (23.9%) than in males (18.5%). While in age group of 30-39 showed the highest prevalence (35.3%) of co-infection. A high prevalence of 47.7% was recorded with CD4+ below 200 cells/μl than 7.6% in participants with CD4+ greater than 200 cells/μl. A highprevalence (49.2%) was also detected in patients with viral load of above 10,000 copies/μl compared to that of those with viral load less than 10,000 copies/μl(12.6%). This study showed a high prevalence of malaria in HIV patients in Awo-Omamma,Oru East, Imo state. This should be considered a great concern to public health. Thus, more effort should be put in research to curb this health issue.
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An investigation into extended spectrum beta-lactamase colonisation in travellers to Laos
More LessIncreased frequency of global travel has facilitated the spread of antimicrobial resistant organisms like extended spectrum β-lactamase (ESBL)- producing organisms and carbapenamase-producing Enterobacteriaceae through increased exposure and transient or persistent colonisation. This study investigates the impact of travel to Laos and of faecal colonisation with ESBLs aiming to help understand the diversity of organisms and also persistency of antimicrobial resistance (AMR) during the study and on return.
Daily faecal samples were collected from 23 doctors visiting Laos over a 21-day period which allowed detection of both constant colonisation and changes to the bowel colonisation over time. Bioinformatic techniques were used to identify ESBL-producing isolates obtained from participants and their contacts. Isolates were sequenced, assembled and annotated. SNP analysis was performed and phylogenetic trees constructed using the core SNP alignment. Escherichia coli was the most prevalent species with a highly diverse array of sequence types. Citrobacter and Klebsiella were the most abundant non-E. coli species. This study confirmed that Laos is an area with high levels of AMR with 28% isolates found to have mobile colistin resistance. There was also a highly diverse and extensive spread of unexpected blaCTX-M genes. Prolonged persistence of resistance genes in the three most prevalent study species found after travellers returned is another serious cause for concern emphasising the extended risk of spread of AMR from high risk to low risk countries. Future work will allow exploration of possible AMR transmission and horizontal transfer of resistance between isolates.
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Defining the RND-binding residues of AcrA
More LessThe resistance-nodulation-division (RND) family of efflux pumps confer clinically relevant antibiotic resistance in Gram-negative bacteria, such as Salmonella enterica. RND pumps, including AcrB, are organized as tri-partite systems, consisting of an inner membrane RND pump, a periplasmic adaptor protein (PAP) and an outer membrane channel. Previously, inactivation of the PAPs AcrA and AcrE in S. enterica has been shown to significantly increase susceptibility to antimicrobials and reduce virulence. Therefore, PAPs are seen as attractive targets for the development of efflux pump inhibitors. However, the role of PAPs in the assembly of tri-partite pumps and the residues involved in PAP-RND pump binding is poorly understood. In this study, point mutations in the predicted RND binding residues of AcrA were generated by site-directed mutagenesis. The point mutants were characterised phenotypically through ethidium bromide efflux assays and antimicrobial susceptibility testing. Furthermore, Western blotting was used to verify that the phenotypic effect of the point mutations was not due to destabilisation of the AcrA protein. Point mutations in certain residues, such as G58, F292, R294 and G363 were found to significantly impair efflux activity and increase susceptibility to various antibiotics and dyes, suggesting an important role for these AcrA residues in RND pump binding. Western blotting confirmed that these point mutants were stable and exhibited similar expression levels to the wild-type. These residues could be important targets for the design and development of PAP inhibitors to restore the activity of existing antibiotics and reduce virulence of Salmonella.
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Food safety knowledge and hygiene practices among the staff of school feeding scheme in the basic schools of Sewoto, South Africa
More LessThe study was to evaluate the food safety knowledge and hygiene practices of the staff of the school feeding scheme in Soweto. A total of 42 food handlers in 13 basic schools under the School Feeding Scheme, Soweto, South Africa were recruited for the study using purposive and convenience sampling methods for the respondents and institutions respectively. A piloted self-administered questionnaire was used. All the respondents were female (100%) with majority being between the ages of 31 and 40 (40%) and had secondary education (63%). About 38 (90.5%) of the respondents indicated that food safety is important, hence, identified “promotion of good health 41 (97.6%), avoidance of bacterial infection 39 (92.9%) and prevention of food poisoning 39 (92.9)” as the major importance. Frequent hand washing 40 (95.2%); cleaning and sanitizing knives/cutting boards 40 (95.3%); checking best before date 39 (92.8); keeping kitchen surfaces clean (80.9%) and checking freshness/appearance of the food upon delivery (88.1%) were indicated as very important food safety and hygiene practices. However, they failed to agree that frozen foods, particularly meat are to be thawed using room temperature (4.8%) and also in the lower shelf in the refrigerators (26.2%) as the best practices. Spearman’s correlation coefficient revealed that no correlation exists between food safety knowledge and hygiene practices. However, there was strong positive correlations among educational level, knowledge and practices (P>0.05). Eventhough they have good knowledge and understanding of food safety issues, they still need training and workshops particularly in HACCP to cover-up the lapses.
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Functional antimicrobial substance and their bioactive compounds extracted from secondary metabolites of Aspergillus terreus
More LessMethods: Fungal isolates were isolated fromsoil samples using Potato Dextrose Agar (PDA). The plates were incubated at 25°C for 72 – 96 h and identification was based on molecular techniques by targeting 18S rRNA. Isolation of bioactive compounds from the extracts was carried out by GC-MS analysis. The extracted secondary metabolites was concentrated by evaporation of the solvents at room temperature. Concentrated extract was constituted by dissolving it in DMSO and stored at 4°C for antimicrobial assay.
Results: Two hundred and fifty six (256) fungal isolates were isolated and sixteen (16) of them showed promising antimicrobial potential. Phylogenetic tree showed evolutionary trend of the fungus with 99% similar to Aspergillus terreus. The broad spectrum activity of the antimicrobial substance were observed to be24.7±4.619 and 26.0±0.000 against Gram positive and negative respectively.The bioactive compounds isolated were phenols, amines, terpenes and fatty acid esters.
Conclusion: The functional and new antibiotics can be obtained from fungal family especially in this era of multi-drug resistant (MDR) bacteria. It is observed that there are functional antimicrobial substances associated with Aspergillus terreus. The extracted substances was found to be active against MDR bacteria in vitro. The bioactive compounds obtained from the GC MS analysis revealed the nature of compounds and some of these compounds have been documented in different areas of antimicrobial and related properties.
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Comparison of proteins expressed during urinary tract infection in young females
More LessIntroduction: Gram-negative bacteria are a major cause of urinary tract infections (UTIs) and particularly Klebsiella pneumoniae (K. pneumoniae), which is a causative agent of 60-70% of community-acquired infections, about 30% of nosocomial UTIs and 20% of recurrent infections.
Materials and methods: Nine urine samples were collected from patients from various clinical departments in King Abdulaziz University Hospital from 2/3/2019 to 2/4/2019. The microbial contents in the urine samples was analysed by urine culture and VITEK analyses. Here, we compared K. pneumoniae proteins profiles to find possible proteins which could shed a light on host-pathogen interactions. The colonies were suspended in a lysing buffer, which then were sonicated, and the proteins contents were separated using 1D SDS-PAGE, analyzed using liquid chromatography-mass spectrometry LC/MS. Proteins showing different expressions in samples were identified by TripleTOF 5600 mass spectrometer.
Results: All of the Klebsiella pneumoniae isolates were ESBL+ and KPC+ as shown on ChromAgar plates. There was no available data on resistance, ESBL or KPC from VITEK2. Hence, ESBL+ or KPC+ data were only obtained from ChromAgar. Additionally, proteomics analysis revealed the following, the total number of different proteins that are expressed from all of the isolates is 2958 proteins. Where in sample U-102, 328 different proteins expressed, 300 different proteins expressed from isolate U-871, 350 different proteins expressed from isolate U-713, in isolate U-755, 378 different proteins were expressed, 207 different proteins expressed from isolate U-754, 305 different proteins expressed from sample U-134, 290 different proteins expressed from isolate U-968, 600 different proteins expressed from isolate U-104, and 200 different proteins expressed from isolate U-659.
Conclusion: The different proteins between the UTI patients indicated specific host pathogen interaction, each isolate expressed different proteins than the other isolate could be reasoned by host pathogen interaction. Host pathogen interaction are influenced by numbers of factor: age, gender, immunity, underling health conditions, antibiotic treatment, acute UTI or recurrent infection all of these factor could have an impact on the type of proteins that are expressed during infection. Even though the isolates are Klebsiella pneumoniae from young females with UTI, they expressed different proteins, these proteins could explain evolutionary development of pathogens and survival in urinary niche, as pathogens need to express certain type of proteins to enable them to live and survival in urinary niche.
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Safety and passivation of faecal contamination in waste
More LessAbsorbent hygiene wastes like nappies and incontinence pads are ubiquitous in municipal and healthcare waste streams around the world as they are convenient products used in child-care and adult incontinence management. Absorbent Hygiene Product (AHP) manufacturing is resource-intensive as the products are required to be of the highest value as they are in almost-constant contact with sensitive body parts. The potential for recovering such valuable resources such as cellulose-based fibres and super-absorbent polymers for reuse in non-food sectors like the construction and wastewater industries has been considered in this study. Appropriate decontamination via chemical methods have been examined using AHPs contaminated with human-associated bacteria.
Findings suggest that for simulated AHP wastes inoculated with 108–109 CFU g-1 of human-associated bacteria like Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pyogenes, a 1:1 ratio of 0.5% calcium hypochlorite/AHP waste is adequate to inactivate the bacteria particularly when combined with an inorganic salt for at least 60 min. Specifically, 4 to 5 log10 reductions were observed. Following such disinfection, material storage and temperatures above 25ºC minimise incidences of microbial regrowth. The disinfection protocol was not found to adversely affect the AHP quality. Overall, such findings suggest that AHP recycling is a potential alternative to current AHP waste disposal practices like incineration (with or without energy recovery) and landfilling.
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The investigation of microbial induced calcium carbonate precipitation for soil improvement
More LessWeak and unstable soils can limit the building of new infrastructure. Current soil strengthening techniques such as chemical grouting have detrimental effects on the environment from greenhouse gas production, soil pH modification and groundwater contamination. Microbial-induced calcium carbonate precipitation (MICCP) is a technique that utilises the ability of bacteria to precipitate calcium carbonate, which can be used for a variety of applications including binding adjacent soil particles and filling the pore spaces of soils to increase their mechanical properties. A commonly used bacterium is Sporosarcina pasteurii. A range of factors influences MICCP which presents challenges with process optimisation. Some studies have made use of computational models to predict biocementation at a larger scale, however aspects of models are based on assumption of conditions instead of experimental data.
An aim of this project is to investigate urease activity in S. pasteurii by comparing different growth media, growth stages, pH and temperatures. Ureolysis kinetics of S. pasteurii will be investigated at different urea and calcium chloride concentrations in liquid media. Finally, the biocementation of S. pasteurii in sand syringe setups will also be investigated to compare the effects of changing influencing factors such as growth stage and cell concentration of S. pasteurii, sand particle size, cementation media concentration, duration between cementation media applications and overall number of cementation treatments. Experimental work will be particularly focused towards gaps in the experimental data used in computational models, to help improve these models and bring MICCP biocementation closer to commercial use.
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Genotypic and phenotypic characterisation of the Klebsiella oxytoca complex
More LessKlebsiella spp. are associated with 3 to 7% of nosocomial infections and can be responsible for a range of conditions including pneumonia, bloodstream infections, meningitis, and necrotizing enterocolitis in infants. The role of Klebsiella pneumoniae in causing disease is well-characterised but, to date, the closely related species Klebsiella oxytoca has not received the same attention, despite often encoding extended-spectrum beta-lactamases and carbapenemases in clinical settings. K. oxytoca is the causative agent of Clostridiodes difficile-negative antibiotic-associated haemorrhagic colitis, a rare condition seen in some individuals receiving antibiotics. Whole-genome sequence analyses have shown K. oxytoca to be a complex comprising at least six species (K. oxytoca, K. michiganensis, K. grimontii, K. huaxiensis, ‘K. pasteurii’, ‘K. spallanzanii’). Our study aims to better characterise the K. oxytoca complex using a polyphasic approach. Preliminary investigations into the genomes of three K. michiganensis clinical isolates revealed the presence of a plasmid-borne ccdABlocus. ccdAB is a toxin-antitoxin (TA) system known to maintain plasmids in other pathogenic enterobacteria. We aim to functionally validate this TA system by cloning and conducting toxicity assays on the CcdB toxin, and cloning and assessing the ability of CcdA to function as an antidote. We also aim to sequence and generate Illumina/Oxford Nanopore hybrid genome assemblies of a larger collection of K. oxytoca complex clinical isolates and investigate their plasmids and TA systems in the same manner.
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The fall and rise of group B Streptococcus in dairy cattle: suspected reintroduction from a human reservoir
More LessStreptococcus agalactiae, also known as group B Streptococcus (GBS), is a pathogen of humans and cattle, in which it is responsible for carriage or invasive disease and subclinical mastitis, respectively. From the 1950s to 1970s, thanks to successful mastitis control programs, the prevalence of GBS fell in the Swedish cattle population, but it re-emerged in the late 1990s. GBS was thought to consist of host-specific subpopulations but recent studies have shown that human and cattle subpopulations overlap, with different accessory genome elements providing survival advantages in each host species. We hypothesized that cattle-adapted GBS was eradicated and replaced by new GBS strains of human origin.
Our aim was to explore the differences in GBS cattle population over six decades (pre-post non-detection), with a focus on the possible role of MGE in the evolution of these strains. Historical (n = 44, 1953 to 1978) and contemporary (n = 76, 1997 to 2012) GBS isolates from bovine milk samples were sequenced and analysed for WGS-MLST. Phylogenetic network analysis revealed the presence of six major clades: two of these were detected only up to 1970, two were only detected after 2004, and two were detected in both periods. Historical isolates were all tetracycline sensitive, whereas 51% of recent isolates harboured tet(M), which is considered a marker of human adaptation. Our data support the elimination of a bovine specific clade (CC61/67) and the emergence of new clades (CC1, CC103/314) that are likely of human of origin.
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Assessing acute and chronic Staphylococcus aureus growth and virulence in an ex vivo model of cystic fibrosis lung infection
More LessStaphylococcus aureus is routinely found in sputum samples obtained from people with Cystic Fibrosis (CF). However, its role in the progression of the disease is unclear. This is important, as antibiotic clearance of S. aureus in CF yields unclear clinical results and there is debate around the utility of anti-Staphylococcal antibiotic treatment. We used an ex vivo porcine lung model (EVPL) to compare the growth and virulence of S. aureus isolates from acute CF exacerbations, with isolates from the same donors when they were stable.
There was no significant difference in mean bacterial load between donors, strains or clinical state. However, when we compared the variance in bacterial load of each pair of exacerbation/stable isolates across experimental replicates of the lung model, we found that stable samples grew more consistently in the EVPL compared to those taken from the same donor during an exacerbation.
Virulence factor assay results were mixed, with results implying greater virulence in either stable or acute samples after passage through the EVPL. We could not detect the AIP quorum sensing signal, which control expression of numerous acute virulence factors, using a reporter assay. We hypothesise that S. aureus might down-regulate Agr expression in the model, consistent with a role as a silent persister, rather than as a pathogenic agent. Further work using the EVPL model will determine how well this reflects the clinical reality in CF.
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Household transmission and host immune evasion factors of LA-MRSA CC398
Staphylococcus aureus uses several strategies to evade the host immune reaction including expression of the genes on the immune evasion cluster (IEC), which target the innate immune response, and tarP, which alters the structure of the bacterial wall teichoic acids to avoid binding of host antibodies. IEC is carried on an ΦSa3 prophage, while the tarP gene is found located on diverse prophages. Livestock-associated methicillin-resistant S. aureus of clonal complex 398 (LA-MRSA CC398) typically lacks the IEC, but a number of Danish isolates have been found to carry the elements regardless.
In this study, whole-genome sequences of 96 isolates from humans and 45 isolates from pigs from the North Denmark Region were used to establish a maximum-likelihood phylogeny from core-genome SNPs and to investigate the prevalence of IEC and tarP. Furthermore, epidemiological and national surveillance data were used to investigate household transmission and the prevalence of IEC in LA-MRSA CC398 isolates from the general population. The study documents several independent acquisitions of IEC on distinct ΦSa3 prophages in humans and an almost 3-fold higher human-to-human transmission rate of LA-MRSA CC398 in households with strains carrying IEC than in households with strains lacking it. No such effect was found for tarP, which is widespread among LA-MRSA CC398 isolates from both humans and pigs. Moreover, IEC also seems to promote spread of LA-MRSA CC398 in the general population. Thus, LA-MRSA CC398 is capable of re-adapting to the human host by acquisition of human-specific immune evasion factors encoded on mobile genetic elements.
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Multidrug and efflux transporters of the model microbe Dictyostelium
More LessThe evolutionarily ubiquitous multidrug and toxin efflux (MATE) proteins mediate anticancer and antibiotic resistance, while transporting toxins, ions and flavonoids in plants. MATEs of the model amoeba Dictyostelium discoideum have not been studied although sequences of its pair group with the two Homo sapiensMATEs. Ddmate1 and 2 are both transcribed, Ddmate2 more so, with peaks in vegetative and slug life-cycle stages. Ddmate1 was upregulated in response to a toxin, ethidium bromide, at the lowest concentration tested. Removing MATE function by inhibitor or mutation increased intracellular levels of various compounds, confirming these as efflux transporters. Plasma membrane localisation was revealed using a GFP-MATE1 reporter-line. MATE1 and MATE2 phenotypes indicated roles beyond detoxification: on Klebsiella lawns these mutants produced significantly smaller plaques than WT, and their axenic growth rates were also lower. The transporters’ impact on use of Dictyostelium for novel drug research was tested using flavonoids. LCMS and fluorescence-imaging revealed differential flavonoid uptake. Flavanones such as naringenin did not cross into cells, whereas flavonols localised to mitochondria and cytoplasm. Ddmate1 transcription was upregulated, however, in response to naringenin, which is known to reduce levels of kidney-disease protein PKD2 in both Dictyostelium and animal cells. Increased flavonol intracellular concentrations confirmed that efflux not import was impeded in MATE1 and MATE2, and kaempferol therefore further reduced MATE1-cells’ growth. These D. discoideum MATEs may usefully model the HsMATEs, aid understanding of flavonoids’ effects, and should be considered when using this model eukaryote to screen drugs.
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Comparison of the distribution and diversity of antimicrobial resistance genes between wild and commercially available Atlantic Mackerel
More LessThe global uncontrolled rise of antimicrobial resistance (AMR) is a major societal threat and it is well documented that AMR is already negatively affecting healthcare and intensive livestock farming systems. Nonetheless, capture fisheries are still essential to the global food supply providing over 50% of the world’s aquatic organism production. To facilitate improved management, it is therefore imperative that we better understand reservoirs of AMR and how gene transmission occurs in the aquatic environment. In order to discern which AMR bacteria and genes are present in the marine environment, sediment and seawater samples were collected and Atlantic Mackerel were captured at the beginning of summer and autumn in the Solent Strait (Portsmouth, U.K). In addition, commercially available Atlantic mackerel were purchased at a local fish market. Using culture-dependent techniques we obtained more than 700 bacterial and 20 fungal isolates from skin, intestinal lining, and intestinal content. Ten different cefotaxime-resistant Pseudomonas spp. were isolated from the seawater and market fish skin samples, and one cefotaxime-resistant Rhanella sp. was isolated from wild fish digesta. Results from ongoing whole-genome shotgun metagenomics analysis will be discussed, as well as the connection between AMR bacteria and AMR gene presence within the marine coastal environment and the local fish markets, which are the last link between capture fisheries and consumers.
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Characterisation of anti-pseudomonad activity of hyper-arid Micromonospora species
More LessThe opportunistic pathogen Pseudomonas aeruginosa is a major cause of nosocomial infections, and has been categorised by the World Health Organisation as a “Priority 1: Critical” target for research and development of novel antibiotics owing to its intrinsic multi-resistance and ability to acquire novel resistance mechanisms.
One strategy for discovering novel antibiotics is the identification and characterisation of metabolites with antimicrobial activity. Members of the bacterial phylum Actinobacteria are historic source of these metabolites, in particular the genus Streptomyces. However, other genera have not received this same level of interest despite sharing the capacity to biosynthesise a diverse array of metabolites. One such genus is Micromonospora, responsible for production of the broad-spectrum aminoglycoside antibiotic gentamicin (M. purpurea).
Here we present three Micromonospora species isolated from the Atacama Desert, Chile; that possess anti-pseudomonad bioactivity inducible by culture on International Streptomyces Project (ISP) Media. In addition, preliminary data indicates that this activity can be affected by the addition of P. aeruginosa conditioned media. In parallel, short-read Illumina sequencing was used to assemble draft genomes for these strains, enabling antiSMASH analysis of putative biosynthetic gene clusters. In addition to this, estimated Average Nucleotide Identity (ANI) as calculated by the autoMLST server indicates that these strains may all be novel Micromonospora species.
The results of this work serve to highlight the biosynthetic capacity of an understudied genus of bacteria, as well as the value of examining underexplored environments and habitats in the search for novel bioactive molecules.
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What are the implications of genetic variation for the control of Japanese encephalitis virus?
More LessJapanese encephalitis virus (JEV) is the leading cause of viral encephalitis worldwide. Annually it causes between 13,000 and 20,000 deaths mainly in South-East Asia. Vaccination programmes have drastically reduced the number of JEV cases however outbreaks do still occur. Recently G1 has overtaken G3 as the dominant genotype in most endemic countries and as all currently licensed vaccines are based on G3 it is necessary to evaluate the potential of emerging genotypes to break through current control measures.
This project seeks to generate a virus-like-particle (VLP) neutralisation assay in order to investigate the potential for emergent or divergent genotypes of JEV to infect vaccinated individuals. Using this assay, we will evaluate the ability of vaccinee sera to neutralise emergent genotypes as well as to introduce a variety of SNPs that have been shown to enhance virulence and investigate whether any combination of these are able to invalidate vaccine protection.
There is also concern that recombination between wild-type and live attenuated vaccine strains may occur in the vaccine setting which could result in rescue of the virulent potential of the attenuated virus. Evidence of this is being sought in silico using publicly available data and existing techniques.
These data will provide a clearer picture of the nature of JEV in endemic areas and whether the current strategy of vaccination is sufficient to prevent naturally acquired infection in the long term or if other strategies must now be considered.
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Characterisation of the antimicrobial mode of action of gallium maltolate
More LessTo human health worldwide. Existing treatments are becoming inefficacious and therefore there is an urgent need for the development of treatments with alternative modes of action. The use of gallium as an antimicrobial agent has been of interest due to its unconventional mode of action involving the inhibition of iron acquisition and metabolism. The structural similarity and inability to reduce from a trivalent to divalent form under normal physiological conditions allows gallium to act as an iron mimetic and inhibit many iron-dependent biological pathways, respectively.
The antimicrobial potential of gallium maltolate (GaM), Ga(III) coordination complex of maltol, was investigated on the opportunistic pathogen Pseudomonas aeruginosa. In vitro and in vivo analyses using Galleria mellonella (greater wax moth) larvae demonstrated the potent bacteriostatic and non-toxic effect of the complex. Subsequent analysis of GaM treated P. aeruginosa via label-free quantitative proteomics provided an insight into the intrinsic mechanisms of action of GaM. Increased expression of iron-storage protein Bacterioferritin B, the HemO component of iron-sulfur clusters and several stress response proteins (Chaperone Proteins ClpB, HtpG and DnaJ) indicate cell stress in response to inhibited iron uptake. Decreased expression of LasA Protease and LasB Elastase quorum-sensing proteins and flagellar motility proteins FlgM and FlgG further demonstrate the growth inhibitory effect of GaM. These findings provide a basis for a better understanding of the mode of action of GaM, a requirement for the improvement of synthesis and efficacy of the treatment.
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Selection and niche trade-offs in biofilm-forming bacterial communities in experimental microcosms
More LessStatic microcosms are a well-established system used to study the adaptive radiation of Pseudomonas fluorescens SBW25 and the adaptive biofilm-forming mutants known as the Wrinkly Spreaders (WS). We have developed this system to investigate selection within multi-species communities using a soil-wash inoculum dominated by biofilm-competent pseudomonads. Here we present community and isolate-level analyses of one serial-transfer experiment in which replicate populations were selected for over ten transfers and 60 days. Although no significant trends in improving community biofilm characteristics or total microcosm productivity were observed, a significant shift in biofilm-formation and microcosm growth by individual isolates recovered from the initial soil-wash inoculum and final transfers indicated that these communities were subject to selection for growth in these microcosms. Surprisingly, the fitness of the archetypal WS was poor when competing against community samples, and having compared the cell densities in the low-O2 region of liquid column below the biofilm, we suggest that part of the community’s fitness advantage comes from the ability to colonise this under-utilised niche as well as to compete at the A-L interface. Samples from the community biofilms and the low-O2 region were able to re-colonize both niches and many final transfer isolates grew throughout the liquid column as well as forming A-L interface biofilms. This suggests that there is a trade-off between fast growth under highly competitive conditions at the A-L interface and slower growth with less competition in the low-O2 region, with some isolates taking a bet-hedging approach a colonizing both niches in our microcosm system.
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The role of air pollution and bacteria in COPD
Air pollution is the single largest environmental health risk worldwide. Particulate matter (PM) air pollution is released as a result of fossil fuel combustion and vehicle motion, breaking and tyre wear. It has been shown that exposure to PM can cause increased levels of respiratory disease, including the exacerbation of COPD, which is frequently associated with bacterial infection. Despite this, the effects of air pollution exposure on COPD associated respiratory bacteria, includingHaemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae are largely unknown. Our recent publication was the first to document that as well as damaging the host, PM has a direct impact on bacteria that can cause respiratory infections. We showed that exposure to black carbon (BC), an important component of PM, results in alterations in biofilm structure in both Streptococcus pneumoniae and Staphylococcus aureus, and increases dissemination of colonising S. pneumoniaein in vivo models.
Following on from this work, we aim to determine how BC impacts the growth, behaviour and virulence of bacteria associated with the COPD exacerbation, including non-typeable Haemophilus influenzaeand Moraxella catarrhalis. Current data show that BC exposure is decreasing the biofilm forming ability of NTHistrains 162 and 375. M. catarrhalis strain M61 biofilm formation is also decreased in the presence of BC, while its growth rate is increased. In addition, pre-exposing NTHi375 cells to BC, prior to infection of A549 cells, increases their ability to adhere to human epithelial cells. This suggests that the frequency of bacterial infection induced COPD exacerbation may be altered in patients from highly polluted areas.
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Investigating the association between organic acids and phenotypic alterations of avian pathogenic Escherichia coli
More LessThe excessive use of antibiotics in agriculture is routinely described as a major contributor to bacterial resistance. Globally, antibiotics are widely used as growth supplements in livestock. This has led to concerns regarding human-use antibiotics in food and food-producing animals. Lately, organic acids (OAs) such as propionic acid (PA) and formic acid (FA) have been increasingly used as alternative antimicrobials or preservatives instead of antibiotics. These are particularly efficient at removing salmonella.
Recently, we have shown that exposure of a Crohn’s Disease associated bacterial pathotype, Adherent-invasive Escherichia coli (AIEC), to PA significantly alters its phenotype resulting in increased adhesion and invasion of epithelial cells and increased persistence through biofilm formation. AIEC are both evolutionarily and phylogenetically related to avian pathogenic Escherichia coli (APEC), however the virulence mechanisms of APEC in poultry remain unclear. The widespread use of OAs as growth supplements and antimicrobials in the poultry industry is a rising concern due to the ability of OAs to alter the bacterial pathotype. In this study, we examined the effect of FA on the phenotype of APEC. We observed that following FA-exposure, APEC showed an increased ability to adhere to and invade human intestinal epithelial cells and form better biofilms both aerobically and anaerobically. Worryingly, these isolates also showed an increased resistance to several antibiotics. These results suggest that the increasing use of alternative antimicrobial such as FA in the poultry industry may lead to APEC strains that are increasingly virulent towards human cells with a potential for increased horizontal transmission
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Dehalogenation of brominated phenolic compounds by environmental microorganisms
Claire Lamb and Joy WattsHalogenated compounds constitute one of the largest groups of environmental pollutants and are a worldwide issue accumulating readily across even remote environments. Contamination by halogenated flame retardants such as polybrominated diphenyl ether (PBDE) is widespread, including marine and freshwater sediments, soil and human tissue. This has been hypothesised to lead to multiple health issues following exposure such as endocrine disruption and immune system alterations. Despite a recent reduction in use, PBDE containing products will continue to leach into the environment for years. Marine sponges are a natural reservoir of brominated compounds and dehalogenation activities of the associated microbiota have been previously observed. It has been postulated that mechanisms for microbial mediated degradation of halogenated compounds also exist within environmental samples commonly contaminated by PBDEs, for example widespread areas of soil sediment. In order to detect and investigate the dehalogenation capabilities of environmental microorganisms, sediment samples from recycling plants have been used to create microcosms with various PBDE congeners (PBDE 47, 99, 153, 209) added, to establish bacterial communities in the laboratory associated with potential PBDE degradation. Once dehalogenation is detected characterisation of debrominating communities from environmental samples will be performed using microbial community 16S rRNA community profiling and QRTPCR. This will provide valuable insight to bacterial communities associated with haloaromatic degradation and further useful information for potential bioremediation of PBDE contaminated soils and sediments.
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Cell surface lipid composition and hydrophobicity governs tuberculosis evolution and pathogenicity
More LessThe evolution of tubercle bacilli correlates closely with changes in cell envelope surface lipid composition (Donoghue et al. Diversity 2017, 9:46; Jankute et al. Scientific Reports 2017, 7:1315). Smooth, hydrophilic “Mycobacterium canettii” is the first recognisable member of the Mycobacterium tuberculosis complex, but it has reduced pathogenicity and poor aerosol transmission. In contrast, rough M. tuberculosis is very hydrophobic and readily spread in aerosols. Starting from hydrophilic surface lipids in environmental Mycobacterium kansasii, intermediate “M. canettii” adds hydrophobic lipids but retains overall cell hydrophilicity. Eliminating hydrophilic lipooligosaccharides (LOSs) and phenolic glycolipids (PGLs) from “M. canettii” leads to M. tuberculosis with a refined selection of hydrophobic lipids, namely phthiocerol dimycocerosates (PDIMs), pentaacyl trehaloses (PATs) and sulfoglycolipids (SGLs). The relative hydrophobicity of M. tuberculosis is double that of representatives of M. kansasii and “M. canettii”.
The above changes have implications both for the onset of tuberculosis and pinpointing evolutionary hosts. Tuberculosis has not been found in Homo sapiens during the Late Pleistocene, but megafauna are the most likely hosts; characteristic bone lesions have been validated by TB DNA amplification and lipid biomarkers in bison metacarpals up to 17,000 years old. Late Pleistocene enhanced TB hydrophobicity and aerosolisation may have produced megafaunal pandemics, with extinction of bison, mastodons and contemporary taxa. The oldest H. sapiens tuberculosis is from the “Fertile Crescent” back to 9-11ka BP at the start of the Holocene. Naïve humans arriving “Out of Africa” may have encountered newly virulent tubercle bacilli of megafaunal origin, recently refined through a distinct “bottleneck”.
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Harnessing microbial science to accelerate the United Nations sustainable development goals
More LessModernisation has thrown humanity and other forms of life on our planet into ditch of problems. Poverty, climate change, injustice, environmental degradation are few of the shared global problems. The United Nations SDGs are set as blueprint to achieve a better and more sustainable future for all. The SDGs are well structured to address the global challenges we face including poverty, inequalities, hunger, climate change, environmental degradation, peace and justice. The SDGs have been driven mainly by international donors and ‘professional’ international development organisations. The world is left with 10 years to achieve these ambitious goals and targets. Various reviews indicated that little has been achieve on overall, and the SDGs will not be reality if new strategy is not in place to bring inclusion. Microbiology, the scientific discipline of microbes, their effects and practical uses has insightful influence on our day to day living. We present how microbiology and microbiologists could increase the scorecard and accelerate these global goals. Microbiology contribution to peace, justice, gender equality, decent work and economic growth will be also highlighted among others. The pledge of Leave No One Behind will fast track progress and microbiology is better position to make this work.
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Intercorrelation mechanism of Hypoxia and Cordycepin Biosynthesis in Zombie fungus
More LessCordycepin is an anticancer metabolite produces by a zombie fungus species of Cordyceps militaris. They are capable to infect and hijack insect’s nervous neuron system. Hypoxic environment commonly must be faced by the pathogenic fungus during infection either this zombie fungus. They activate oxygen sensing mode, heme, siderophore, and sterol biosynthesis to overcome it. Underlined our previous study that liquid surfaced culture of C. militaris NBRC103752 produced a higher amount of cordycepin than submerged culture, suggesting that hypoxic conditions might induce it. However, when and how the mechanism of cordycepin production started in liquid surfaced culture is not understood, yet. In our present study, the combination of transcriptomics and gas chromatography-mass spectrometry were carried out during the production phases of cordycepin (5d, 12d, and 19d of incubation periods) and the mechanism of cordycepin production was figured out. The expression of genes in the fermentation pathway and the oxidative phosphorylation pathway were significantly upregulated and down regulated, respectively. Expression of four genes in the heme biosynthesis, including 5-aminolevulinic acid synthase (CCM_01504), delta-aminolevulinic acid dehydratase (CCM_00935), coproporphyrinogen III oxidase (CCM_07483) and cytochrome c oxidase15 (CCM_05057) were upregulated at the beginning of the exponential phase (12d). Further, the activation of Zn(2)-C6 transcription factor that regulates the iron acquisition and ergosterol biosynthesis significantly upregulated and a metabolite reporter adenosine was detected only at 12d. The results in the present study show the correlation between hypoxia and the accumulation of heme before cordycepin biosynthesis.
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