- Volume 1, Issue 1A, 2019
Volume 1, Issue 1A, 2019
- Poster Presentation
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- Infection Forum
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The impact of a horizontally acquired virulence plasmid on Bacillus cereus G9241, the causative agent of an anthrax-like illness
More LessBacillus cereus, Bacillus anthracis and Bacillus thuringiensis, are Gram-positive, spore- forming bacteria and principle members of the Bacillus cereus sensu lato complex. The species are highly similar at a chromosomal level, but are phenotypically diverse due to the presence of different plasmids. The anthrax pathogen, B. anthracis contains two virulence plasmids, pXO1 and pXO2. The pXO1 plasmid carries the anthrax toxin genes which are involved in intracellular survival and suppression of immune cell function while pXO2 carries capsule genes which are required for the pathogen to evade phagocytosis. Both plasmids are required to allow B. anthracis to act as a highly virulent mammalian pathogen. As well as encoding toxins, the pXO1 plasmid encodes atxA, a transcriptional regulator that is able to control gene expression from both the plasmid and chromosome. It is proposed that AtxA is incompatible with the chromosomally encoded PlcR, a global transcriptional regulator which controls expression of secreted haemolytic and cytolytic toxins. This has led to the genetic inactivation of plcRin all B. anthracis isolates and driven the evolution of high mammalian virulence. Interestingly, there are several B. cereus isolates that possess a pXO1-like plasmid, called pBCXO1, which are capable of inducing an anthrax like illness. Importantly, genome sequencing of one such strain, B. cereus G9241, revealed intact copies of both atxA and plcRgenes. This project aims to understand how G9241 has evolved to accommodate both regulators. We have used a pBCXO1-cured strain of G9241 to study the influence of AtxA and pBCXO1 on the biology of G9241 and how it interacts with human macrophage.
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A liposomal drug delivery system for improved eradication of Helicobacter pylori
More LessHelicobacter pylori is the leading cause of peptic ulcers and gastric cancer. Eradicating H. pylori infections is becoming more difficult due to increasing antibiotic resistance and poor patient compliance. We aim to develop a novel liposomal drug delivery system that encapsulates antibiotics and the antimicrobial fatty acid linolenic acid (LLA). We hypothesise that H. pylori will have much lower resistance rates to this dual formulation. Liposomes consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), sphingomyelin, and cholesterol were produced using the thin film hydration method. The size of our liposomes was assessed using dynamic light scattering (DLS), and their antimicrobial activity was assessed using a viable count assay. The liposomes had a particle size ranging from 95 to 150 nm. We have successfully loaded LLA up to 20 % of total lipid composition, and shown stability of the particles in storage at 4 °C for up to 3 months. The LLA liposomes achieved complete eradication of H. pylori in vitro at 150 µg ml−1, whereas control liposomes containing no LLA had no antimicrobial effect. We have recently formulated liposomes encapsulating LLA and amoxicillin at 2.2 mg ml−1 and 0.99 mg ml−1 respectively, and are currently assessing their antimicrobial activity against H. pylori. In conclusion, it is possible to prepare LLA- and amoxicillin-containing liposomes and LLA has antimicrobial activity against H. pylori. In future work we will utilise strategies to enhance gastric retention which will provide local bactericidal activity, improving on inefficient systemic uptake mechanisms with current therapies.
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Isolation and characterization of Clostridioides difficile spores from contaminated single-used surgical gowns
More LessClostridium difficile is the primary cause of antibiotic associated diarrhoea globally. In the UK there has been a decline in the prevalence of C. difficile due to implementation of surveillance and infection control procedures. At Rideout Hospital, USA, however, there is a high incidence of C. difficile infection, which has been partly attributed to poor infection control measures. Other factors include the ability of spores to adhere to fomites such as surgical gowns. It has been demonstrated that the single-use polypropylene surgical gowns used at Rideout can ‘trap’ hydrophobic epidemic spores of C. difficile within the fibres, which can then be transferred to stainless steel surfaces and hospital floor vinyl; even with use of appropriate sporicides such as sodium dichloroisocyanurate. This study sought to establish the strains of C. difficile present on the gowns and thus inside the nosocomial environment. Contaminated gowns from Rideout were cultured for 5 days anaerobically in Brain –Heart Infusion broth supplemented with 0.1 % Sodium taurocholate. Broth culture was screened for the presence of C. difficile using CCFA media, C. DIFF QUIK CHEK COMPLETE®, 16 s-23s RNA analysis and toxin PCR. Once isolated, strains were sequenced and tested for biocide susceptibility to in-use concentrations of Sodium dichloroisocyanurate. In total 23 suspected C. difficile samples were isolated from the gowns; of which 8 were confirmed. Sporicide susceptibility testing is ongoing. Once infective strains have been identified measures can be taken to enforce appropriate infection control procedures in order to limit the prevalence of spores and reduce infection rates.
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Understanding the ecology and evolution of polymicrobial wound infections
More LessChronic wounds (CW) are a common complication of diabetic ulcers (DUs), which are a major burden to health care systems worldwide and can result in lower limb amputation due to the intractability of the infection. In DUs there is a high probability of the infecting bacteria evolving considerable phenotypic and genetic diversity, as has previously been shown for chronic lung infections. However, it is not known whether this is also the case for chronic DUs, and whether diversity impacts on virulence and antibiotic resistance. To study this, bacterial populations were isolated from different samples from patients with DUs. Phenotypic diversity was investigated in P. aeruginosa populations through the analysis of phenotypes traditionally associated with pathogenicity, and through a whole genome study. Phenotypic variation in P. aeruginosa isolates taken from different patients was observed, but little variation within the same CW (with exception of one patient with a leg ulcer). Antibiotic resistance was found to increase during the course of infection, and it became apparent that P. aeruginosa colonisation in DUs is via a single strain per ulcer, and potentially per patient, even though some sample-specific phenotypic profiles were found to arise from a homogenous population. For this case, a detailed genomic analysis between bone and blood isolates was done, including a comparison of their transcriptomes using RNAseq. The results suggest that the loss of flagellum facilitated evasion of the innate immune system, which allowed bacteria to go undetected and spread systemically causing the rapid decline in the patient’s health.
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Incidence of Pseudomonas aeruginosa resistance in a tertiary hospital
Antibiotic is a formidable remedy to infections caused by diverse microbial agents. This assertion is however questioned in the wake of antimicrobial resistance. Fifty clinical isolates of Pseudomonas aeruginosa were obtained from both in and out-patients using standard procedure. The isolates were identified using standard biochemical tests. The antibiotic susceptibility pattern of each isolate was examined inaccordance to the Clinical and Laboratory Standards Institute (CLSI) guidelines using the Kirby-Bauer’s disc diffusion method. The antibiotics used in the study includes: Ciprotab, Colistin-sulphate, Meropenem, Ceftraxone and Cefepine. Out of the clinical isolates obtained, a total of 48 per cent male and 52 per cent females were the population under study. The percentage ratio of in-patient and out-patient examined were 32% to 68 %. The percentage distribution of the administration class for medical and surgical was 34% and 66% respectively. The highest incidence of Pseudomonas aeruginosa was from patients that have undergone cesarean section (28%). Highest susceptibility was observed in Ciprotab (82%) Meropenem (64%) and Ceftraxone (46%). Highest number of resistance was observed against Cefepine and Colistin Sulphate while less than 5 % were resistant to Ciprotab and Meropenem. Meropenem and ciprotab were the two classes of drugs that showed highest activity against Pseudomonas aeruginosa. Commonly used antibiotics must be continuously examined for its efficacy. Anti-microbial susceptibility monitoring is necessary inorder to guide physicians in prescribing the right combinations of anti-microbials to limit and prevent the emergence of multi-drug resistant strains of P. aeruginosa.
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No clinical benefit of empirical antimicrobial therapyfor pediatric diarrhea in a high-usage, high-resistance setting
More LessPediatric diarrheal disease presents a major public health burden in low- to middle-income countries. The clinical benefits of empirical antimicrobial treatment for diarrhea are unclear in settings that lack reliable diagnostics and have high antimicrobial resistance (AMR). In this study, conducted a prospective multicenter cross-sectional study of pediatric patients hospitalized with diarrhea containingblood and/or mucus in Ho Chi Minh City, Vietnam. Clinical parameters, including disease outcome and treatment, were measured. Shigella, nontyphoidal Salmonella (NTS) and Campylobacter were isolated from fecal samples, and antimicrobial susceptibility profiles were determined. Statistical analyses, comprising log-rank tests and accelerated failure time models, were performedto assess the effect of antimicrobials on disease outcome. Among 3166 recruited participants (median age 10 months; interquartile range, 6.5–16.7 months), one-third (1096 of 3166) had bloody diarrhea, and 25 % (793 of 3166) were culture positive for Shigella, NTS, or Campylobacter. More than 85 % of patients (2697 of 3166) were treated with antimicrobials; fluoroquinolones were the most commonly administered antimicrobials. AMR was highly prevalent among the isolated bacteria, including resistance against fluoroquinolones and third-generation cephalosporins. Antimicrobial treatment and multidrug resistance status of the infecting pathogens were found to have no significant effect on outcome. Antimicrobial treatment was significantly associated with an increase in the duration of hospitalization with particular groups of diarrheal diseases. Our results imply a lack of clinical benefit for treating diarrhea with antimicrobials in a setting using high antimicrobials; adequately powered randomized controlled trials are required to assess the role of antimicrobials for diarrhea.
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- Irish Fungal Society Clinical Case Studies
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Comparison of methods for detection of Candida in bronchoalveolar lavage (BAL) of cancer patients
More LessAn accurate and rapid identification of Candida species in cancer patients with pulmonary symptoms can provide important information for effective treatment. Candida infections represent an increasing cause of morbidity and mortality in patients receiving immunosuppressive chemotherapy for cancer, organ transplantation or in immunocompromised. We used conventional methods such as culture, fermentation reactions, morphology and molecular methods based on the ribosomal DNA repetitive regions or the Internal Transcribed Spacer (ITS) for the identification of Candida species. Seventy bronchial specimens of Bronchoalveolar Lavage (BAL) from cancer patients at Shaukat Khanum Memorial Cancer Hospital and Research Center. Lahore, Pakistan were included in this study. Seventy cancer patients were diagnosed on the basis of histological profile. Candida detected by conventional methods using Sabouraud Dextrose Agar (SDA), potassium hydroxide (KOH) preparation, germ tube test, fermentation reactions and Gomori methanamine-silver stain (GMS). Thirty (42 %) positive isolates of Candida species were obtained by culture, twenty (28 %) isolates were germ tube test positive while thirty isolates (42 %) were positive by PCR method. In conclusion, the results of our study showed that the PCR based detection methods are significantly better and can detect Candida with more accuracy and specificity as compared to conventional methods. Our study would pave the path for optimization of protocols for detection of Candida in cancer patients.
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Film-forming agents as potential barriers to fungal skin infections
More LessBackgroundSuperficial fungal infections are one of the most common causes of human disease caused by dermatophytes or yeasts. Dermatophyte infections are caused by fungi that can digest keratin, infecting the keratinised tissues e.g. skin, hair and nails. It has a higher prevalence than the other superficial mycoses, and its incidence has increased continuously over the last few decades, probably because of the change in lifestyle and frequent usage of antibiotics. Therefore, the aim of the project is to develop a physical barrier that can prevent the early stages of infection to the skin, to avoid development of antifungal resistance and cross-contamination.
Materials/methodsWe developed an ex vivo model using porcine skin to study the potential of film-forming agents in prevention and treatment of dermatophytosis caused by Trichophyton rubrum. We used cell viability assays, confocal and electron microscopy to study the effects of film-forming agents on T. rubrum, followed by using QTOF-LCMS and NMR to analyse the carbohydrates binding and chelation to study its mechanism of action.
ResultsA cationic polymer used in pharmaceutical and cosmetic products inhibited growth of T. rubrum on porcine skin. Viability assays indicated that the polymer has a fungistatic activity and microscopy imaging indicated it formed a coating on top of T. rubrum. The QTOF-LCMS and NMR indicated the polymer inhibits fungal growth by removing the carbohydrate content and chelation.
ConclusionsThe present study suggests this cationic polymer has considerable antifungal activity against Trichophyton rubrum by preventing the supply of nutrients to the fungi.
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Probing the role of histone modifications in the evolution of pathogenicity in Candida glabrata
More LessCandidiasis is one of the most prevalent mycoses worldwide, and there are few successful treatments for this disease. In the UK, Candida glabrataaccounts for 25 % of Candida infections, and due to the increasing incidence of multidrug resistance, C. glabrata poses an eminent threat to public health, exacerbated by the limited range of antifungal therapies. Our aim is to elucidate the molecular mechanisms underlying the emergence of pathogenicity in the C. glabrata lineage, to identify novel therapeutical targets. Using comparative phylogenomics, we putatively identified 19 genes under positive selection in the C. glabrata lineage. Each of these genes influences chromatin structure by regulating histone post-translational modifications (PTMs). To assess the contribution of these genes to virulence, we monitored the phenotypic consequences of individually removing each gene in clinically-relevant assays. To date, we have focused on 3 genes, cg-SPP1 (regulates histone H3 methylation), cg-HAT1 (histone H4 acetyltransferase) and cg-AHC1 (subunit of Ada histone acetyltransferase complex). Preliminary data show that the individual deletion of these genes increases biofilm formation and fluconazole resistance in C. glabrata. Furthermore, acetyltransferase knockout strains show hypervirulence in an in vivo Galleria melonella infection model, and phenotypic differences in abiotic stress assays. RNA-sequencing was performed on the type-strain, cg-hat1Δ, and cg-ahc1Δ, in the presence and absence of fluconazole to determine the molecular bases for these phenotypes, revealing commonality between differentially expressed genes in both mutants. Together, our data suggest that histone PTMs play a significant, and overlapping, role in dictating virulence in C. glabrata, and detail the global transcriptomic response to fluconazole.
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Candida albicans TLOs and fitness: phenotypic analysis of a TLO null strain of C. albicans generated via CRISPR-Cas9 mutagenesis
More LessBackgroundCandida albicansis a fungus that is both a commensal and opportunistic pathogenic in humans. Genomic analysis has highlighted the expansion of the telomere associated ORF (TLO) gene family as unique in C. albicans. In C. albicans there are 15 different members of the TLO family present, compared to only two in its closest relative, C. dubliniensis. Here we show that deleting all TLOs from C. albicans reduces fitness and effects multiple phenotypes.
MethodA guide RNA (gRNA) with sequence with homology to the TLOgenes was introduced to C. albicans AHY940 (aLEU2/Δleu2), along with the other components of the CRISPR-Cas9 (Nguyen et al. 2017).
ResultsThe Δtlo strain was found to be generally pseudohyphal in morphology, compared to the wild type. It also showed defective growth in nutrient rich YEPD and YEP-Galactose. Growth on YEPD agar produced colonies similar in appearance to WT, however hyphal induction via growth on Spider agar was greatly reduced in the mutant strain compared to WT. Resistance to oxidative stress was examined and the Δtlostrain was more susceptible to stress induced by both H2O2 and tBOOH. In tests to determine the resistance of the Δtlostrain to cell wall perturbating compounds it was seen that this strain is much less resistant to Congo Red and Calcofluor white than WT. Biofilm formation on plastic surfaces also was reduced in the Δtlo strain compared to that of the WT.
ConclusionsDeletion of the TLO genes in C. albicans greatly impacts phenotypes associated with virulence, and generally results in a less fit strain of C. albicans.
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- Marine Protists as Emerging Models for Functional Genomics and Cell Biology
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Development of a genetic modification system for the dinoflagellate Amphidinium carterae
More LessDinoflagellates are important marine algae, being an essential symbiont in corals. Loss of the dinoflagellates causes coral bleaching, and ultimately death of coral reefs. Current efforts to study dinoflagellates are greatly hindered by the lack of a reliable method of genetic transformation. The chloroplast genome of dinoflagellates is fragmented into multiple plasmid-like minicircles, each carrying at most a few genes. We have used ‘artificial’ minicircles, based on fusions between endogenous minicircles and E. coli plasmids to establish a transformation system and optimize parameters for it. We have determined the sensitivity of wild type dinoflagellate strains to possible selective agents. We have compared the performance of a number of transformation methods, including electroporation and particle bombardment, and find that electroporation is not effective for transformation, whereas particle bombardment (‘biolistics’) is. We have assessed different parameters for biolistics and subsequent selection. We have shown the successful maintenance of sequence from an artificial minicircle within a dividing cell population over a period of six months, as well as evidence (using RT-PCR) of transcription and the expected phenotype for the inserted gene. This represents a significant step forward in developing the genetic modification of Amphidinium carterae. We are currently testing the protocol with other dinoflagellates.
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The unorthodox chromosomal organisation of the dinoflagellates
I Ian Hu and Ross WallerDinoflagellates nuclei are unlike any other. They have: 1) highly inflated genome sizes, 2) practically lack histones and nucleosomal organization; 3) have permanently condensed liquid crystalline chromosomes throughout the life cycle, and; 4) contain a novel a major new nuclear DNA-binding protein. This protein, called Dinoflagellate/Viral NucleoProtein (DVNP) is small in size (10–20 kDa), highly positively charged (30–40 % R+K), and its gene is one of the most highly transcribed in dinoflagellate cells. It has no homology to histone proteins, has no homologues in either eukaryotes or prokaryotes, but is found in a number of marine large DNA viruses. To understand the role of DVNP in the dinoflagellate nuclei we have expressed and purified DVNP and are studying the properties of this novel protein and its interaction with DNA. We show that DVNP is a monomer in solution, but upon exposure to DNA it rapidly binds to and compacts DNA into complexes micrometers in size. Using single-molecule imaging and optical tweezers, DVNP is seen to compact DNA a rates of over 50 µm/sec and change the mechanical properties of DNA. Most interestingly, the DVNP/DNA aggregates show a propensity to travel along the DNA strand en masse. Together, these observations suggest that DVNP plays a central role in the novel model for chromatin management found in dinoflagellates.
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- Microbial Dark Matter
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Phylogenetic and genomic characterisation of narnaviruses: a diverse group of non-encapsidated RNA viruses
More LessNarnaviruses are a group of single-stranded, positive-sense RNA viruses, which are non-encapsidated and hence normally transmitted vertically. Narnaviral genomes encode a single protein: the RNA-dependent RNA polymerase (RdRp), which catalyses viral replication. Currently, there are just two recognised species within the genus Narnavirus, both of which are known to infect Saccharomyces yeast. Here, we systematically identify and characterise narnaviral genomes in public sequence databases, using a combination of in silicoapproaches. We identify two major clades of narnaviruses, and propose the establishment of a taxonomic framework based upon their molecular characteristics. Codon usage bias across both clades was analysed and compared with those of potential host taxa from across the eukaryotic domain of life. In one clade, we demonstrate the widespread presence of a long reverse-strand open reading frame (rORF), which typically occupies >90 % of the full-length genomic RNA. Comparative analysis shows that the putative rORF-encoded proteins are highly divergent in amino acid composition, with a central region of increased conservation. These findings shed new light on one of the most divergent clades of eukaryote-infecting viruses.
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Comparative analysis of the composition and change of the microbiome of diabetic foot ulcers from patients on different therapies
More LessDiabetic foot ulcers (DFUs) are slow healing wounds which arise from co-morbidities associated with diabetes. Often these ulcers become infected leading to gangrene, osteomyelitis and sepsis. Current treatment options include debridement and a topical irrigant which have limited success. Understanding the effects that these treatments have on microbiome of DFUs and on wound healing is poorly understood. This study compared the efficacy of two irrigant solutions (Prontosan and Electrolysed water –E.W.) on their impact on the DFU microbiome, their role in DFU healing and their effect on biofilm viability. Sequential samples taken from 7 patients undergoing treatment with either one of the irrigants, over a 4 week period revealed commonly observed genera present included Staphylococci (96 %), Propionibacterium (96 %) and Finegoldia (89 %). A unique composition and diversity was observed in the microbiome of each individual DFU. Increasing microbial diversity within the DFUs was correlated with an elevated percentage abundance of anaerobic and Gram negative genera whilst inversely correlated with facultative anaerobic and Gram positive genera. No significant reduction in diversity or species richness of the DFU microbiomes was observed after treatment with either irrigant. Both Prontosan and E.W. had similar effects upon S. aureus biofilms reducing viability by 82.013 % and 86.89 % respectively however E.W. efficacy was strain specific. In addition, E.W. was ineffective at preventing biofilm formation in 6/8 (75 %) S. aureus strains. Better understanding of the DFU microbiome and investigations into novel therapies is paramount to aid our ability to improve the quality of life for diabetic patients.
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Characterising the human intestinal mycobiome during healthy ageing
More LessAlthough fungi are fundamental to the human microbiome, the diversity and dynamics of the mycobiome is poorly understood, particularly in considering their association with infectious disease, autoimmune disorders and atopy that affect immunocompromised individuals and infants. Characterising the human mycobiome faces several challenges relating to their low abundance and lack of standardized procedures for sample collection and isolation of viable cells and/or quality genetic material for culture-dependent and independent taxonomic and functional characterisation. To address these issues, we have developed a mycobiome analysis pipeline employing both culture-dependent and independent methods to identify as well as isolate, where possible, the fungal taxa populating the human intestinal tract. In a proof-of-concept study this pipeline has been used to identify fungal populations in faecal samples obtained from a small cohort of young infants, aged 2 years or younger. All were born prematurely, and severely immunocompromised and at risk from invasive and potentially lethal microbial infections, including those caused by fungal overgrowth. We have used this combined approach successfully to identify the fungi present in each individual infant, and to recover viable isolates. To date, Candida albicans and C. parapsilosis are the most frequently isolated fungi. While both are major opportunistic human fungal pathogens, C. parapsilosis is particularly problematic to preterm babies, due to its innate ability to form biofilms. Detailed characterisation of these isolates is currently underway. Two large-scale longitudinal microbiome studies have started at the Quadram Institute, and our validated analysis pipeline will be incorporated to define the fungal component of each study participant.
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Extraction and identification of components of the biofilm matrix in Pseudomonas species biofilms
More LessThe surface associated communities of microorganism in biofilms are encased in a matrix of extracellular polymeric substances (EPS). The EPS is made up of mainly polysaccharides, proteins, nucleic acids and lipids and plays an important role in maintaining the integrity of the biofilm (1). Although the general composition of the EPS is known, it can be highly variable among strains and among different growth conditions for the same strain. Due to the large variety of biopolymers in nature and the difficulty in their analysis, EPS has been called ‘the dark matter of biofilms’ (2). In order to develop a comprehensive understanding of the matrix of the biofilm, EPS was extracted from four Pseudomonas spp., mCherry-expressing Pseudomonas fluorescens, GFP-expressing Pseudomonas putida and the wild types of Pseudomonas fluorescens and Pseudomonas putida. The extractions were carried out on biofilms grown on glass slides using the cation exchange resin (CER) method. Colorimetric methods were used to quantify the sugars and proteins present in the EPS. These colorimetric assays showed that there was a larger amount of proteins present compared to sugars. The proteins present in all four biofilms of Pseudomonas spp. were identified by LC-MS/MS while NMR and HPLC were used to identify the sugars present. The knowledge gained by these results have the potential to aid in the development of biofilm eradication methods through the targeting of specific components of the EPS. 1. Starkey, M., et al. (2004), American Society of Microbiology: 174–191.2. Flemming, H.-C. and J. Wingender (2010). Nature Reviews Microbiology 8 : 623.
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Fundamental nanoparticle interactions with biofilms of Pseudomonas species
The use of engineered nanoparticles (NPs) as a technique for antimicrobial delivery aimed at biofilm treatment is an emerging field of research. There have been numerous studies involving a wide range of NPs showing varying results regarding anti-bacterial effects (1). However, research focusing on specific interactions between functionalized nanoparticles and the extracellular polymeric substances (EPS) of biofilms are limited. The complexity of the biofilm matrix may be hindering the understanding of the fundamentals which govern biofilm – nanoparticle interactions. There are a wide range of physicochemical properties which influence the uptake and retention of nanoparticles within the matrix including NP size and charge properties, biofilm topography and porosity and EPS composition (2). These aspects must be considered when studying biofilm – nanoparticle interactions. In order to identify these specific interactions, a series of experiments were carried out using mCherry-expressing Pseudomonas fluorescens and GFP-expressing Pseudomonas putida biofilms. Using high throughput fluorescent intensity measurements and confocal microscopy, it was possible to investigate the uptake of surface functionalized silica NPs by the two biofilms and obtain valuable information regarding biofilm – nanoparticle interactions. The results suggest that specific NP surface functionalization has a major role in guiding the interaction and binding of EPS components, possibly due to electrostatic interactions between NPs and the EPS. The findings of this research will help with the future design of nanoparticles with specific modes of action towards components in the EPS.1. Ramos M., et al. (2018). International Journal of Nanomedicine13 : 1179–1213.2. Nevius BA., et al.(2012). Ecotoxicology21 : 2205–2213.
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Shining a light on microbial dark matter: a role for the forgotten B vitamins in marine algal communities?
More LessThe ocean is home to a huge diversity of life that perform a wide range of functions key to earth systems. Marine algae communities contribute a significant proportion of net carbon fixation of the globe as well as forming the basis of the food webs for every trophic level above their own. Despite their importance, many of the taxa that make up these microalgal communities are relatively unknown and understudied, due to their recalcitrance to lab culturing. This concept is known as microbial dark matter and applies in particular to many non-photosynthetic lineages which have been long overlooked by the research community. To investigate whether dependencies of these taxa on certain B vitamins may play a role in this unculturability, data sets produced form the Tara oceans expedition will be analysed. The Stramenopile lineage will be analysed in greater detail for their metabolic potential to synthesise the B vitamins, and/or whether they may dependent on an external source. Both Single-Amplified Genomes (SAG) and metatrascriptomic data sets will be analysed to determine the complement of genes present in different Stramenopile lineages. This will then be correlated with the global distribution data attainable from analysing Tara data sets. By this means, we hope to understand more about the metabolic contribution of this taxonomic group to the community, how this supports the community as a whole and whether dependence on B vitamins is a contributing factor to unculturability.
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Investigation into the physicochemical interactions of silica nanoparticles and EPS biomolecules within the biofilm matrix of Pseudomonas spp.
Difficulties in the removal of bacterial biofilms in the industrial and biomedical sectors have driven the development of new technologies. Although numerous studies have highlighted the use of nanoparticles (NPs) as antibiofilm agents, the fundamental physicochemical interactions between NPs and the biofilm matrix is still poorly understood 1. The development of ‘smart nanoparticles’ for biofilm removal requires an in-depth understanding of the complex interactions between NPs and biomolecules within the extracellular polymeric substances (EPS) of the biofilm matrix. These interactions are highly dependent on the physical and chemical properties of the NPs 2. In order to identify and characterize the specificity of binding and the direct interaction between silica NPs (SiNPs) and EPS matrix components of Pseudomonas spp. biofilms, a range of experiments were carried out. Biofilms were exposed to SiNPs of different sizes, charges and surface functionalization while biomolecules such as proteins, polysaccharides, and eDNA were fluorescently labelled and their distribution, relative abundance and their colocalization with SiNPs within the biofilm was quantitatively assessed using CLSM microscopy. Changes to the SiNPs size and surface-chemistry dramatically affected their interactions with biomolecules in the biofilm matrix. This includes the increased affinity (or interaction) of SiNPs to preferentially bind to proteins and beta-linked polysaccharides and also lead to changes in the degree to which aggregation of SiNPs occurs within and on the surface of the biofilm.
1. Ikuma K et al. (2015). Front. Microbiol. 6, 591
2. Bewersdorff, et al. (2017). Int. J. Nanomed. 12, 2001–2019
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Leveraging structural relationships as a novel mode of viral classification
More Less100 years have passed since the independent discovery of the humble bacteriophage (phage) by Frederick Twort and Felix d’Herelle in 1915 and 1917 respectively, and since then, it has become commonly accepted that phages represent the most abundant biological entities on Earth. Despite this fact, viral taxonomy lies in extremely treacherous waters, ever changing to accommodate the next series of phylogenetic mysteries. The utilisation of genes such as the terminase large sub-unit can in some cases provide a robust taxonomic marker, but this is often found to fail at higher taxonomic levels. In addition, the rapid evolutionary dynamics and highly modular nature of phages provide yet more phylogenetic roadblocks, necessitating additional and multifaceted approaches as a means of resolution. Here, we describe a novel approach towards the taxonomic classification of phage systems. Tools for accurately predicting the three dimensional structure of proteins are improving at an unprecedented rate due to the fact that the number of protein sequences far exceeds the number of experimentally determined structures. Our approach leverages these methods through a pipeline which compares models of phage marker genes in order to permit the inference of phylogenetic relationships based on cross model superimposition. We hope this method will supplement other approaches in providing a more holistic approach to viral classification.
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Characterisation of the microbiome for two hexactinellid sponges and purification of associated antimicrobial agents from their resident microbes
The imminent threat of antimicrobial resistance has necessitated that the search for novel antimicrobials be widened to lesser-explored environments. Marine and freshwater sponges have emerged as the most prolific source of such compounds over the last decade, representing the most widely sampled phyla in the hunt for novel biologics over the last 45 years. Most of the work however has focused on sponges from shallow waters, with the deep-sea sponge microbiome highlighted as a major source of untapped antimicrobial potential. Optimisation of bacterial recovery was carried out for two previously unstudied species of deep-sea Hexactinellid sponge species (Pheronema carpenteri and Rhabdodictyum delicatum recovered from the Rockall Trough, North Atlantic), using a variety of culture media, supplementation and environmental conditions. This optimisation was carried out in parallel with 16S rDNA metagenomic sequencing in order to determine community composition for both sponge species (IonTorrent, Life Technologies). All recovered isolates were assayed for antimicrobial activity, forming a panel of ‘active’ organisms. Two isolates (Ph16-28; A11) were selected for downstream purification and characterisation of the responsible antimicrobial agent via column chromatography. Isolate identities are currently being confirmed via draft whole-genome sequencing (MinION, Oxford Nanopore), and are suspected to be members of the Bacillus and Streptomyces genera. Current data provides a working axiom for the cultivation of deep-sea sponge microbes and suggests the deep-sea sponge microbiome to be a promising source for novel antimicrobials.
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Uncovering the dark matter of the metagenome one read at a time
Contemporary metagenomic annotation methods have proven insufficient in our attempts to better understand the complex environments around us. We call the yet to be annotated part of a metagenome it’s ‘dark matter’. The Gene Ontology (GO) is a hierarchical vocabulary used to describe gene product function and a large collection of curated genes with GO annotations already exists. DeepGO utilises deep learning to build models from these curated genes and gene products to predict GO categories for novel proteins. One of the major problems with metagenomic studies today is the process of assembling the environmental DNA sequences into their original genomes. This is difficult, with chimeric metagenomically assembled genomes being common. To avoid this and the computational and time expense, we have modified DeepGO to perform protein function prediction directly from sequence reads with limited protein coding sequence prediction. Three independent models were trained as the following; The first 50 amino acids of a protein were used for training, The last 50 amino acids were used for training, A phasing window of 50 amino acids was used to train across the entirety of a protein sequence. These models were chosen to learn from the different parts of a protein sequence we are likely to capture from only the short unassembled sequence reads. We compared the three models by producing a mock metagenomic community consisting of 6 model bacterial genomes. We evaluated the functions predicted from the unassembled sequence reads and the protein coding sequences predicted from the assembled metagenome.
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Effect of dietary olive oil and palm oil on rumen bacterial composition in dairy cows
More LessThe rumen bacteria play a major role in lipid metabolism. Bacteria remove the double bonds of unsaturated fatty acids resulting in the production of saturated fatty acids, which are incorporated in milk. Crude olive oil (rich in unsaturated fatty acids) represents a potentially valuable feed source for dairy cows that might enhance the human-health beneficial composition of milk and dairy products. This project studied the effect of supplementing dairy cow diets with olive oil (OO) and palm oil (HVO) on rumen microbiota. For 63 days the animals were fed a control diet (basal diet) with no added lipid and two fat-supplemented diets (30 g kg−1 DM). Rumen sampling were performed at the onset of the experiment and every 21 days for 63 days using a rumen scoop. Total microbial DNA was extracted from ruminal samples for high-throughput sequencing of the 16S rRNA gene through Illumina MiSeq platform. Results revealed the dominance of phyla Firmicutes and Bacteroidetes. Firmicutes was the most prevalent phyla in diet control (75.2 %), OO (71.1 %) and HVO (75.2 %). At genus level Succiniclasticum and Prevotella were the dominant genera, belonging to Firmicutes and Bacteroidetes respectively. Succiniclasticum decrease significantly their relative abundance during OO supplementation (p)
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- Microbial Physiology, Metabolism and Molecular Biology Forum
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Studies on amylase from protoplast fusants of Aspergillus species using response surface methodology
More LessImproved amylases were developed from protoplast fusants of two amylase-producing Apergillusspecies. Twenty regenerated fusants were screened for amylase production on Remazol Brilliant Blue agar. Crude enzymes were produced by solid state fermentation on rice bran were assayed for activity. Three variable factors (temperature, pH and enzyme type) were optimized for amylase activities of parent and selected fusants on a rice bran medium by solid state fermentation. The variables assessed were optimized using the Central Composite Design (CCD) of the Response Surface Methodology (RSM). Amylase activities at room temperature and at 80 °C showed Aspergillusdesignates, T5 (920.21 U ml−1, 966.67 U ml−1), T13 (430 U ml−1, 1011.11 U ml−1) and T14 (500.63 U ml−1, 1012.00 U ml−1) as preferred fusants. Amylases produced by the fusants were observed to be active over the range of pH studied. Fusants T5 and T14 had an optimum acidic and alkaine pH respectively. Optimization studies revealed enzyme T5 at pH 4 and temperature of 40 °C as optimum for amylase production. The statistical tools employed, predicted and compared the optimal conditions for enzyme activities of amylases from parent and fusant strains of Aspergillus revealing the desirability of the fusants over the parents in industrial applications.
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Sero-epidemiology of scrub typhus among suspected cases in selected areas of Nepal
More LessIntroductionScrub typhus is an acute, febrile, infectious disease which is caused by Orientia tsutsugamushi. In the current days of Nepal huge burden of scrub typhus have been found, mainly Southern Nepal.
MethodologyBlood samples were collected from the suspected patients of scrub typhus who is having acute febrile illness. Detection of Immunoglobulin M (IgM) antibody to Orientia tsutsugamushi was performed by using Scrub Typhus Detect™ kit, In Bios International USA.
ResultsA total 1585 cases, 358 (22.58 %) were positive for IgM Antibodies to Orientia tsutsugamushi. Multivariate analysis demonstrated that the following factors were significantly associated with the scrub typhus. 1. Females (odd ratio [OR]= 2.037, P=< 0.001, confidence interval [CI]= 1.465 – 2.831) 2. Rural residential location (odd ratio [OR]= 0.431, P=0.001, confidence interval [CI]= 0.261 – 0.714), 3. House near grassland (odd ratio [OR]= 3.279, P=<0.001, confidence interval [CI]= 1.932 – 5.563), 4. Presence of mouse inside the house (odd ratio [OR]= 5.462, P=<0.001, confidence interval [CI]= 4.048 – 7.371), 5. Working in the field (odd ratio [OR]= 9.845, P=0.004, confidence interval [CI]= 2.068 – 46.954).
ConclusionThe study indicated that Scub typhus is a big burden of Nepal, where we have identified the prevalence rate was 23 % . Use of IgM ELISA test will help for early diagnosis and it is urgent to investigate to save the life of people who lives endemic areas of Scrub typhus in Nepal.
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Alterations in the gut microflora balance of neonatal wister rats induced with aflatoxin M1 in milk
More LessAflatoxin M1 is a metabolite of the most potent aflatoxin, (AFB1) and thus, has been treated and rendered not so toxic, on this basis, its study has been taken for granted. It is the aim of this study to ascertain the toxic nature of AFM1 by determining its effects on microbial flora in the gut of neonatal rats. A dosing experiment was conducted on the neonates, where they were divided into groups and treated with different concentrations of AFM1 using uncontaminated milk as a carrier medium into the rats. The rats were sacrificed; the small and large intestine were harvested and cultured on appropriate selective media for growth of microorganisms. Results show samples from the control group had an uninterrupted microbial community, while the treated group, with increasing doses of AFM1 decreases and depletes the microflora in the gut samples. Lactic acid bacteria were also significantly depleted by AFM1. These findings suggest the capability AFM1 in modifying the gut microbiota in a dose-dependent manner which might result in serious health hazards in neonates.
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The role of acetyl-phosphate in the pathogenesis of Neisseria gonorrhoeae
More LessAcetyl-phosphate (AcP), an intermediate from the phosphotransacetylase-acetate kinase (PTA-AK) pathway, has shown to be critical in pathogenic bacteria for the general metabolism and synthesis of virulence factors. Lysine acetylation is a post-translational modification (PTM) that occurs enzymatically and non-enzymatically by the addition of an acetyl residue from acetyl coenzyme A and AcP, respectively. Neisseria gonorrhoeae, the etiologic agent of gonorrhoea, has been shown to use AcP for lysine acetylation, however, the role that AcP has in the pathogenesis and how acetylation is regulated has not been discerned. The concentration of AcP was altered in N. gonorrhoeae MS11 by interrupting the genes involved in the PTA-AK pathway, pta and ackA, and the gene that encodes for a lysine deacetylase family protein, kdac. AcP concentrations were increased in ΔackA and decreased in Δpta resulting in modulation of lysine acetylation. Growth on glucose, lactate or pyruvate were investigated. In aerobic conditions, ΔackA mutant solely grew in glucose, while the Δpta mutant grew in glucose and lactate. In microaerophilic conditions, ΔackA and Δpta mutants solely grew in presence of glucose. The virulence of ΔackA and Δpta was tested by infecting larvae of Galleria mellonella. WT killed 50 % population (n=15) after 6 days and ΔackA after 24 h, however, Δpta after 6 days it only killed 10 %. Taken together, our results show AcP as an important metabolite for the metabolism and virulence of N. gonorrhoeae.
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Biofertilizer/biopesticide potentiality of zinc solubilizing Pseudomonas aeruginosa FA-9 and Enterobacter sp. FA-11 isolated from the wheat rhizosphere grown in arid zone
More LessBacterial strains were obtained from wheat rhizosphere and screened for zinc solubilization on agar plates. Two strains FA-9 and FA-11 were found efficient for Zn solubilizing activity and were identified as Pseudomonas aeruginosa and Enterobacter sp. by 16S rRNA and gyrase (gyrB) genes analysis, respectively. The strain FA-9 produced a clear zone diameter of 63 mm, 60 mm and 51 mm on agar plate amended with different zinc ores. The strain FA-11 produced a zone diameter of 17 mm with zinc carbonate and 20 mm with zinc oxide while no zone was observed with zinc phosphate. Both strains showed no visible activity with ZnS ore. Similarly, FA-9 and FA-11 increased maximum soluble zinc content (102 µg ml−1 and 45 µg ml−1) from zinc carbonate ore as compared to zinc oxide ore (102 µg ml−1 and 45 µg ml−1) in liquid broth. It was noted that both strains exhibited less potential (7 µg ml−1 and 0.57 µg ml−1) to solubilize ZnS ore in liquid broth. A comparison between agar plate assay and liquid broth quantification shows that agar plate assay does not present the solubilizing potential of ZSB precisely. The strains FA-9 and FA-11 produced auxin with l-tryptophan (3.25 µg ml−1 and 2.86 µg ml−1) and without l-tryptophan (1.23 µg ml−1 and 1.02 µg ml−1). Both strains expressed exo-polysaccharides (EPS) and siderophores activity along with phosphate (P) solubilization, ACC deaminase and antifungal activities. The ACC deaminase and N-fixation activity was confirmed by the amplification of acdS and nifH genes respectively.
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Proteomic analysis of Escherichia coli associated with urinary tract infections
More LessUrinary tract infection (UTI) is considered to be one of the most prevalent bacterial infections in the world. It affects urinary tract system including bladder and kidney. Uropathogenic Escherichia coli is more prevalent in females due to their anatomical structure as well as they are more susceptible to recurrent infections. Every woman out of three is affected by UTIs. Gram-negative bacteria are a major cause, particularly Escherichia coli (E. coli). E. coli was considered a main causative agent for 80–90 % of community-acquired infection and for about 40 % of nosocomial UTI. Moreover, it is responsible for 25 % of recurrent infection. Proteomic can be used to analyze and identify complete components of proteins. It can be used to distinguish between bacteria based on synthesized proteins. In addition, proteomic is applicable to identify possible targets of therapy. My study aims To compare protein profiles of E. coli from different UTI patients and identify possible unique protein signature for future biomarker studies. Six Urine samples with E. coli were taken from females aged from 15 to 50 years old. Samples loaded onto Sodium dodecyl sulfate Polyacrylamide gel electrophoresis for separation. Samples with abundant proteins profiles on SDS-PAGE, were selected for run on two dimensional gel electrophoresis. Gels were compared to each other to look for interesting protein spots. Many differences were observed in protein profiles of E. coli isolates in both 1D SDS-PAGE and 2DGE. Two bacterial proteins identified as possible candidate were (OmpA) found in Gram negative bacteria and RNA polymerase-binding transcription factor DksA mostly found in E. coli
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Cyclic di-nucleotides – what is their role in biofilm formation and pathogenicity of Fusobacterium nucleatum?
IntroductionCyclic di-nucleotides (CDNs) act as important second messengers in bacteria, regulating multiple cellular functions, including biofilm formation. Fusobacterium nucleatum is a key player in disease-associated biofilms in periodontitis (gum disease). Previous studies revealed the importance of CDNs in the virulence of other dental pathogens such as Porphyromonas gingivalis, but their function in F. nucleatum virulence remains elusive. Here, we aim to elucidate their importance in the pathogenicity of F. nucleatum.
MethodsUsing bioinformatics, we identified a putative dual adenylyl/guanylyl cyclase in the genome of F. nucleatum ATCC 23726, the only currently genetically tractable strain. Consequently this target gene was deleted from the chromosome. Wild-type and mutant strains were grown in single- and multi-species biofilms, the amount of biomass quantified by crystal-violet assay and the biofilm topography analysed using scanning electron microscopy. Additionally, intracellular CDNs were quantified using LC-MS/MS.
ResultsDifferences in biofilm formation comparing wild-type, mutant and further F. nucleatum subspecies will be presented. Furthermore the level of CDN production of those strains will be shown.
ConclusionsDisease-associated biofilms in periodontitis affect over 50 % of the adult UK population. The disease can be debilitating, potentially leading to tooth loss and the bacteria involved have also been associated with systemic diseases such as cardiovascular disease, arthritis or certain types of cancer. Understanding the involvement of CDNs in the pathogenicity of F. nucleatum and its interaction with various periodontal pathogens might provide new insights into prevention and treatment of periodontitis and other conditions.
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Carbon, Nitrogen and Phosphorous assimilation in V. cholerae – a case of imperfect nutrient sensing
More LessVibrio cholerae experiences frequent feast and famine conditions. In the absence of all the three nutrient components (Carbon, Nitrogen, and Phosphorous), the cells could manage starvation and survive up to 6 months with only 1 log reduction in viability. Addition of carbon or nitrogen to the starvation media resulted in 3 log reduction in cell number. Simultaneous addition of carbon and nitrogen (Phosphorous starvation) reduced the cell viability to a below detectable level. The suboptimal growth conditions; non-metabolizable source of carbon and nitrogen; variable C: N ratio; inhibition of metabolism and cell division, increased the cell survival under phosphate starvation. However, when all the three components are limited, the cells did not initiate active metabolism and could conserve the energy for long-term survival. This observation suggests that integration of carbon, nitrogen and phosphorus sensing is imperfect in V. cholerae and it cannot down-regulate the metabolism during phosphorous limitation. The carbon and nitrogen could prime the cells to accelerate the rate of metabolism, irrespective of the presence of phosphorous, thereby creating an energetically unfavorable situation. The lack of crucial component phosphorus fails to activate a stringent response that results in increased futile cycling of nutrients, loss of ATP and cell death. The Vibrio genus was found to be less efficient in surviving phosphate starvation than E. coli and S. Typhimurium. The two-component system CreC (a response regulator of phosphorous) is absent in V. cholerae and may be responsible for the lack of stringent response to phosphorus starvation.
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Glucose metabolism via the Entner-Doudoroff Pathway in a select subgroup of Campylobacter jejuni
More LessCampylobacter jejuniis is the most common cause of bacterial gastroenteritis worldwide. It is often considered a ‘commensal’ in chickens where it rapidly colonises the caecum of young chicks and is present in around 80 % of farmed poultry. C. jejuni has a small genome and most strains are unable to transport or metabolize glucose. Instead they use amino acids, TCA cycle intermediates and short chain fatty acids as energy and carbon sources. Recently, a WGS project identified an unusual group of C. jejuni (RG-2 group) within a bank of C. jejuni strains isolated from farm associated Norway rats. These strains had acquired an entire locus (glc) of seven genes which enable uptake and metabolism of glucose via the Entner Doudoroff (ED) pathway. This project is addressing the impact of these genes on metabolism and niche survival of these strains. Campylobacter are microaerophilic bacteria. Good growth of C. jejuni NCTC 11168 and of aglcnegative Norway rat C. jejuni isolate was recorded in the presence of 5 % O2up to 13 %in a Whitley M35 workstation. In contrast, two glcpositive strains of C. jejuni, Dg275 and Dg95, exhibited the unusual ability to grow well in an atmosphere of 16 % oxygen, as monitored by c.f.u. and OD600. Current studies are also focusing on the contribution of glucose utilization via the ED pathway to influence survival and growth in oxygen.
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CRP i.e. cAMP receptor protein provides a competitive edge to Salmonella Typhimurium in a microbial community set-up
More LessThe survival of enteric pathogens in the multi-nutrient environment involves interaction with other organisms. The fact that competition for nutrient resources has shaped the networking of metabolic pathways is well known. The cAMP receptor protein i.e. CRP is a keystone regulatory protein, connecting various metabolic pathways. Our aim was to study the importance of CRP in nutrient uptake and utilization in Salmonella Typhimurium under intra-species and inter-species nutritive competition. The crp gene knockout (Δcrp) was co-cultured with the wild-type or other pathogens. The Δcrp failed to compete with the wild-type Salmonella Typhimurium, Escherichia coli, Vibrio cholerae and Staphylococcus aureus in nutrient intensive media. However, the survival of the co-cultured Δcrp was unaffected in nutrient-poor media. These results suggest that CRP is necessary for the effective acquisition of readily available nutrients as found in rich media in co-culture. The role of released antimicrobials or surface proteins of the co-cultured strains was also overruled by culturing the mutant in the supernatant of these organisms and separating the cultures in the same media respectively. The co-cultured Δcrp showed an enhanced survival when overall metabolism was reduced with low temperature and antibiotics like chloramphenicol. A circumstantial evidence that CRP manages the global and limits the futile metabolism is provided by this study. The absence of CRP doesn’t affect the survival of the standalone culture. However, the role of this protein becomes obvious in a bacterial community setup. Therefore, CRP is crucial for Salmonella to survive in an intestinal and external environment.
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Molecular studies on iron metabolism, redox stress and pathogenicity in Bartonella
More LessFor bacteria, mechanisms of resistance to redox stress are utilised to increase survival. Although B. henselae has the ability to resist redox stress, its genome sequence is characterised by a paucity of genes responsible for redox-stress resistance, particularly hydrogen peroxide degradation systems. However, our results show a surprisingly high resistance to peroxides, given the lack of peroxide disposal systems. To determine how B. henselae achieves resistance to H2O2 stress, the potential role of MbfA (membrane-bound bacterioferritin), which in other α-Proteobacteria is believed to function as an iron exporter, was investigated. The results show that B. henselae has the ability to export iron and that this export activity is promoted by H2O2as export was inhibited by exogenous catalase and anaerobiosis. The form of iron exported was largely ferric. The impact of the iron export process on the resistance to, and degradation of, H2O2, by B. henselae was determined and the results showed that B. henselae mediates a rapid consumption of exogenously supplied H2O2. This degradation was entirely inhibited when iron chelators were included along with the H2O2. The resistance of B. henselae to NO was also tested since NO is generated by phagocytic host cells along with H2O2, and is suggested to potentiate the toxicity of H2O2 towards engulfed bacteria through inhibition of haem-dependent catalases and alkylhydroperoxidases. Our results suggest that NO does not cause a marked increase in H2O2 toxicity for B. henselae, in contrast to E. coli (a haem-catalayse/peroxidase dependent bacterium).
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Disruption of the mce operon from Streptomyces affects spore resistance and results in precocious germination
More LessStreptomyces coelicolor is a non-pathogenic soil saprophytic bacterium and is a model organism for antibiotic production. This species contains a single copy of a nine gene cluster known as the mammalian cell entry (mce) operon. This operon was originally characterised in Mycobacterium tuberculosis as an important virulence factor acting in invasion and survival within macrophages and encodes an ABC transporter for cholesterol import. As the function of the mceoperon in S. coelicoloris currently unknown, this study aims to characterise the operon through deletion of the mcelocus and resulting impact on bacterial morphology and survival. SEM images demonstrate that spores of a mcedeletion mutant (Δmce) display a wrinkled, and ‘fragile’ phenotype, with spores appearing to germinate whilst on the spore chain. Heat kill assays show that the deletion of the mce operon result in S. coelicolor spores which are less tolerant to temperatures of 60, 70, 80, 90 and 100°C compared to WT S. coelicolor spores. Heat activation of Δmce spores was also consistently absent at all temperatures tested. The spores of a Δmce mutant also exhibit a precocious germination phenotype seen on SEM images confirmed with germination assays.
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Metabolic recuperation in valine production by mutant strain
More LessThe contemplation of present research work to get over scantiness of amino acid by amelioration ofmicroorganism. As a matter of fact the use of chemical and physical factors symbolize a new era in the excerption of microorganism for proficient of producing a desired product. In this workbiosynthetic pathway of valine and their regulation was observed in Bacillus cereus, Corynebacterium and Pseudomonas fluoresence. The contrivance of operation of ems mutants was analyze in different fermentation media at 24, 48, 72 and 96 h incubation under optimum condition by acidic ninhydrin method. Optical density was recorded by spectrophotometer 470 nm for Valine. The effects of carbon and nitrogen sources and growth factors on the production of valine were studied. In this studies mutant evince that mutation confabulate upon the bacteria the ability to produce valine many times more than its parental strain within the same span of incubation period. The objective of this work to step up headway to search unconventional sources to overcome nutritional exhortation of valine.
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Biochemical screening approach to identify regulatory DNA-binding proteins
More LessThe interaction between transcription factors, promoter elements and RNA polymerase is crucial to bacterial adaptation to physical and chemical changes in the environment. Transcription factor discovery has strongly relied on the isolation of regulatory mutants followed by biochemical confirmation. However, highly pleiotropic regulatory mutations can often be deleterious, unstable and difficult to select, or could directly or indirectly affect gene expression at multiple levels. Here, we describe a biochemical approach to identify trans-acting regulatory proteins independent of their cellular function that could be used as an alternative to genetic screens. The method consists in (i) incubating bacterial lysates with an immobilized DNA encompassing a promoter as defined by RNA-Seq data (ii) pull down of bound proteins and (iii) liquid chromatography tandem mass spectrometry (LC/MS/MS). We tested the usefulness of this approach by identifying proteins binding to the Vibrio cholerae rpoS promoter that drives the expression of the general stress response regulator RpoS. The approach identified several proteins binding to the rpoS promoter that included the factor for inversion stimulation (FIS), and the master quorum sensing regulator HapR. Binding of both purified proteins to the rpoS promoter was confirmed by electrophoresis mobility shift assays. The role of fis and hapR on rpoS expression was examined in strains containing a chromosomally-integrated rpoS-lacZ fusion. Deletion of fis had little effect while HapR appeared to enhance rpoS expression.
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Electrophotonics: multimodal sensors for bacteria identification and phenotyping
More LessA comprehensive understanding of bacteria phenotypes requires tools that are able to characterise structure and function across multiple length scales, from communities and individual cells down to single molecules. Multimodal sensing combines multiple transduction technologies in parallel to probe different properties simultaneously, and thereby increase the range of measurable interactions, the amount of information that can be extracted, and improve detection accuracy. Electrophotonics is a multimodal sensing approach that combines electrochemical and photonic techniques in a single, integrated device that provides enhanced quantitative measurements of chemical reactions. We present a new electrophotonic device based on a Si3N4 guided mode resonant (GMR) structure with an integrated indium tin oxide (ITO) electrode. The GMR structure is sensitive to refractive index changes at the sensor surface, enabling label free, real time detection of biomolecules, microorganisms and imaging of molecular interactions with micrometre-scale resolution to provide spatial information about surface binding interactions. The ITO electrode has been shown to be compatible with voltammetry-based techniques for interrogating redox behaviour along with electrochemical impedance spectroscopy. We demonstrate the wide range of microbiological applications of electrophotonic technology including the characterisation of redox active protein electron transfer and surface adsorption, bacterial adherence and growth on chemically functionalised surfaces, and label free parallel detection of clinically relevant biomarkers. We believe that the multimodal measurements with this novel technology can provide new approaches to investigate and understand microbial biology.
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D-serine: trick or treat?
More LessD-serine is an amino acid that has become a focus in recent years due to its unique role in many biological processes. It is a host metabolite in humans with diverse roles in neurotransmission and signalling. Previous work in our group showed that D-serine can play a critical role in controlling expression of pathogenic virulence factors in bacteria, specifically Escherichia coli, as well as impacting microbial community composition through niche specificity. In enterohaemorrhagic E. coli (EHEC), the presence of D-serine results in down regulation of the type 3 secretion system (T3SS), resulting in inability to colonise. In contrast, uropathogenic E. coli (UPEC) can catabolise this metabolite and colonise the bladder where D-serine is present in high concentrations, leading to a urinary tract infections (UTI). Hence, in different pathotypes, D-serine can act as a positive (treat) or negative (trick) environmental stimulus. UPEC and neonatal meningitis E. coli (NMEC) strains often carry the DsdXCA operon which allows for the metabolism of D-serine. This locus is responsible for the detoxification of D-serine, allowing for tolerance of D-serine as a carbon source. My work has focused on understanding the role of D-serine as a signal for gene expression in UPEC and NMEC through the action of the regulator, DsdC. Using global approaches I have characterised the binding sites of DsdC across the chromosome revealing new insights into how this protein contributes to UPEC and NMEC pathogenesis. The work is important as it helps us understand how specific pathogens sense their environment and cause disease.
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Enigma of gastric microbiome: diversity, genomic and transcriptomic insights into Ochrobactrum spp. (a non H. pylori, urease positive bacterium)
More LessThe dominance and pathogenicity of Helicobacter pylori in the gastric niche is eminent. Urease test is used to diagnose the infection of H. pylori. However, nowadays several reports indicated the emergence of organisms other than H. pylori (Non H. pylori bacteria– NHPs). We have reported urease positive, Ochrobactrum intermedium from the non-ulcer dyspeptic individual. Therefore such urease positive bacteria raise a question on the reliability of urease test. Since 2005, nearly 35 cases are reported for the presence of Ochrobactrum spp. from clinical specimens. Their high level of resistance to antibiotics and phylogenetic relationship to anthropozoonotic pathogen, Brucella makes them a probable human pathogen. Therefore, it is necessary to study their prevalence, survival mechanism and function in the human stomach. To check their pervasiveness in the Indian population, 218 urease positive gastric biopsy samples were processed and 62 Ochrobactrum spp. isolates were identified with and without H. pylori. Population study of Ochrobactrum spp. was also done using multilocus sequence typing (MLST) which revealed 45 sequence type and clonal population. As the prevalence of Ochrobactrum spp. is approx. 30 %, their survival mechanism was studied under in vitro gastric conditions (acidic pH, urea, microaerophilic environment), using microarray and RNA seq methods. The differential gene expression analysis showed 2 different acid resistance mechanism, i.e. Urease dependent acid resistance mechanism like H. pylori, and Amino acid dependent acid resistance mechanism. Our finding necessitates further detailed investigation of such NHPs in gastric environment and role of such bacteria in gastric niche and warrants further refinement of urease based diagnosis.
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Novel nitrogen-containing heterocyclics with bactericidal activity against Mycobacterium tuberculosis
Tuberculosis (TB) is the leading cause of global morbidity and mortality caused by an infectious disease, with over 10 million new cases emerging in 2017. This global emergency is exacerbated by the emergence of multi-drug and extensively-drug resistant TB, therefore new drugs and new drug targets are urgently required. From a whole-cell phenotypic screen we identified a series of nitrogen-containing heterocyclic compounds that elicit potent anti-mycobacterial activity with MIC values <10 µM against Mycobacterium tuberculosis. Interestingly, this series of compounds demonstrate no detectable drug resistance in mycobacteria. Mode of action and target deconvolution studies suggest that these compounds inhibit mycobacterial growth by interfering with late-stage mycolic acid biosynthesis. In addition, these compounds exhibit a suitable toxicological and PK/PD profile that paves the way for further development as an anti-TB chemotherapy.
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Expression, purification and antimicrobial activity of recombinant pediocin PA-1 M31L, a PA-1 derivative with enhanced stability
Pediocin PA −1 is class IIa bacteriocin that displays efficient antimicrobial activity against pathogenic Listeriaspp. This bacteriocin is known to lose activity during long periods of storage especially at non optimal pH, thus reducing its usefulness for the pharmaceutical and food industries. Loss of activity has been attributed to oxidation of the methionine residue at position 31, however, replacement of this residue by leucine results in a peptide with activity equivalent to that of the native peptide. In this work, the heterologous expression of the structural (with Met31 to Leu substitution), accessory and transport genes from pediocin PA-1 operon was carried out in Escherichia coli TunerTM (DE3) cells. The sequences of all genes were redesigned using codon bias for the host and were cloned into an expression vector that allows control of plasmid copy number. The heterologous expression of pediocin Met31Leu was optimized for temperature, induction time, IPTG concentration and plasmid copy number and was evaluated via antimicrobial activity assays against Listeria innocua DPC3572. Maximum activity (2560 AU mL−1) was achieved using low plasmid copy number and 6 h of induction at 37 °C with 1 mM of IPTG. Recombinant pediocin PA-1M31L was successfully purified in 5 steps (>95 % purity) as confirmed by mass spectrometry (4606.27 Da) with a yield of 0.725 mg per liter of culture. This variant showed a similar spectrum of activity to the native pediocin PA-1 and is an interesting alternative for industrial applications due to its greater stability.
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A possible alternative for chromosome dimer resolution in E. coli
More LessDimer formation is a serious threat to the stable maintenance of ColE1-like plasmids. Dimers form infrequently by homologous recombination but accumulate rapidly by having two origins of replication. This results in elevated plasmid loss and a reduction in host cell growth rate. Plasmid dimers are resolved to monomers by the XerCD recombinase plus accessory proteins ArgR and PepA, acting at the cer recombination site. The circular chromosome of E. coli also forms dimers infrequently, and consequent failure of chromosome partition results in filamentation, SOS induction, and failure of cell division. Site-specific recombination is required for dimer resolution during cell division in a process facilitated by XerCD acting at the dif (deletion induced filamentation) site near the E. coli chromosome terminus. ArgR and PepA accessory proteins are nonessential, but the septum-associated protein FtsK is necessary for dimer segregation, suggesting the XerCD/difcomplex interacts with division septums. Our preliminary work had revealed homology between cer and a 170 bp chromosomal site (tcs, terminal region cer-like site) 1.2 min from dif. The tcs site and surrounding region was cloned into plasmids, dimer plasmids were purified and then assayed for tcs-mediated recombination. Our results demonstrate that a construct with a 500 bp tcs insert supports XerCD-mediated recombination, whereas smaller constructs were recombination-deficient. The absence of plasmid monomerization in mutant strains indicates that ArgR and PepA are required for recombination. Additionally, the tcs knockout strain displayed moderate filamentation of cells. Given the similarities between cer, tcs, and dif, these results suggest that tcs could facilitate chromosome dimer resolution.
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Post-transcriptional regulation of gene expression in Escherichia coli experiencing sustained nitrogen starvation
More LessWhile transcriptional reprogramming is perhaps the most well understood form of controlling gene expression in response to nitrogen starvation in bacteria, how post-transcriptional regulation (PTR) of gene expression contributes to this adaptive response remains elusive. Small regulatory RNAs (sRNAs) are the major post-transcriptional regulators of gene expression in bacteria. They regulate gene expression by base pairing to target mRNAs, leading to enhanced translation or inhibition of translation and/or alteration of mRNA stability. To form productive interactions with target mRNAs, most sRNAs require an RNA chaperone. In many bacteria of diverse lineages, the RNA chaperone Hfq plays a central and integral role in the PTR of gene expression by stabilising sRNAs and promoting their interactions with cognate mRNAs. Comparative analysis of the transcriptomes of Escherichia coli at different stages of nitrogen starvation reveal that levels of sRNA vary throughout starvation. We used Hfq as a surrogate to study sRNA-mediated PTR of gene expression during sustained nitrogen starvation. Our results indicate that sRNAs-mediated PTR of gene expression plays a major role in the adaptive response to sustained nitrogen starvation. Intriguingly, using single-molecule PALM, we reveal that Hfq is involved in the formation of intracellular structures which functionally might resemble processing (P) bodies found in eukaryotic cells involved in mRNA turnover.
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Functional characterisation of cell wall proteins that enhance nasal colonisation by Staphylococcus aureus, through binding to host mucin
More LessHuman nasal colonization with Staphylococcus aureus sets the stage for subsequent systemic infection. The mechanisms responsible for colonization are not fully understood. This study characterizes S. aureus adhesion to nasal mucosa in vitro and observes the interaction of S. aureus with mucin. S. aureus showed significantly higher binding to mucin during stationary phase in comparison to log-phase cells. Adherence of S. aureus srtA mutant to mucin was not significantly different from wild-type, thus Srt A shows no influence on S. aureus interaction with mucin. Adherence to mucin was saturable in a dose- and time-dependent fashion. Biotin–labelled mucin bound to surface protein (55 kDa) of cell wall extracted S. aureus which is encoded by the sbi gene. These data suggest that adherence factors are present on the surface of S. aureus such as sbi. Purified recombinant Sbi was prepared and the mucin binding capacity of the protein was tested by ELISA. In order to determine the function of specific domains of Sbi in adhesion, Sbi constructs with, without the IgG-domain and with B2-glycoprotein domain were expressed on the surface of E. coli BL21(DE3). The expression of Sbi with the B2-glycoprotein domain on the surface adhesion, emphasizing the role of Sbi in mucin adhesion. A profound binding effect was observed with Sbi incubated in wells coated with host mucin. Therefore, it is proposed to investigate a novel interaction of S. aureus to host mucin in order to control S. aureus nasal colonization.
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Identification of essential residues for polyprenol phosphate mannose synthase and protein O-mannosyl transferase activities required for protein glycosylation in Streptomyces
More LessActinobacteria have a protein O-glycosylation system that resembles eukaryotic protein O-mannosylation. Both M. tuberculosis and S. coelicolor have growth retarded phenotypes when protein-O-mannosyl transferase (Pmt), which transfers mannose from polyprenol phosphate mannose to a target protein, is absent. Moreover, S. coelicolor pmt- mutants are resistant to φC31 phage infection and have increased susceptibility to vancomycin and multiple b-lactams. S. coelicolor strains that lack polyprenol phosphate mannose synthase (Ppm1), which transfers mannose from GDP-mannose to polyprenol phosphate, are even more susceptible to antibiotics and a ppm1- mutant in M. tuberculosis is lethal. Pmt and Ppm1 are therefore possible new targets for the isolation of antimicrobials to be used against M. tuberculosis. Our aim is to further understand the structure and function of these enzymes. Sequence alignments and structural bioinformatics were used for S. coelicolor Ppm1 and Pmt to identify site-directed mutagenesis targets. Mutant alleles were introduced into ppm1- (DT3017) or pmt- (DT2008) S. coelicolor strains using conjugative integrative plasmids and scored for their ability to complement phage sensitivity and antibiotic hyper-susceptible phenotypes. Twenty-two highly conserved Pmt residues were each changed to alanine and six mutants failed to complement DT2008, indicating essentiality. Modelling these six residues indicated that five are positioned close to the predicted catalytic DE motif. For Ppm1, ten mutant alleles were tested and eight were essential for DT3017 complementation, with four residues positioned close to the predicted catalytic DXD motif. Whilst some of the mutations were predicted to impair catalytic activity, others may have affected localisation or substrate binding.
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Intrageneric competition of staphylococci reveals discrete evolutionary outcomes
More LessWe previously identified the diversity of antimicrobial activity in nasal microbiomes that correlates with presence and absence of Staphylococcus aureus. The major competitor of skin surfaces is Staphylococcus epidermidis that can express diverse antimicrobial activities. Ten-fold less frequently found in skin and nasal microbiomes, Staphylococcus hominis is relatively unstudied. The aims of the research were to develop insights into the factors of S. hominis that contribute to the dynamics of competition with these major skin colonising staphylococci. One prominent inhibitory strain, S. hominis was selected to culture with both S. aureus SH1000 and S. epidermidis with aim to use genome sequencing to reveal loci contributing to competition. S. aureus evolved during the competition of S. epidermidis and inhibitor-producing S. hominis to reveal two discrete phenotypes. Non-pigmented clone expressed less alpha-haemolysin and had a SNP in agr Cencoding the receptor of the Agr quorum-sensing system. More Pigmented clone had a SNP in sig Bencoding the accessory sigma factor required for expression of the staphyloxanthin. Distinctively, competition of S. aureus SH1000 with S. epidermidis revealed evolution of S. aureus that corresponded with a SNP in the lytSgene of the LytSR two-component system controlling murein hydrolase activity and autolysis. Pacbio genome sequencing followed by use of AntiSMASH revealed the S. epidermidis inhibitory strain, which enabled persistence of S. hominis, encoded the lantibiotic gallidermin biosynthesis operon on a 39 kb plasmid. Gallidermin decreased S. aureus survival to competitor S. epidermidis, and an increased ability of S. hominis to maintain its population size during evolution experiments, which supported the dynamic relationship of the three staphylococci. Future studies will unravel explanations for the contributions the identified loci make to multi-species competition.
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A multiplatform approach to investigate the structure and architecture of the biofilms of Pseudomonas aeruginosa and Staphylococcus aureus in response to antimicrobial treatment
More LessBacterial biofilms are highly complex communities, composed of highly structured extracellular polymers and subpopulations of differentiated cells, such as persisters. These contribute to the resistance of bacterial biofilms to antibiotics, creating a significant issue in the treatment of infections and resulting in elevated levels of mortality and morbidity. Here, we use a microfluidic system coupled with time-lapse microscopy and fluorescent dyes for exopolysaccharide (EPS) and extracellular DNA (eDNA) to investigate how the architecture of the growing biofilm is ordered. We show that in Pseudomonas aeruginosa EPS is deposited first in the initial stages of microcolony development, but that eDNA then acts as a leading edge for further microcolony expansion. We explore how this assembly is perturbed by the introduction of different antimicrobial agents. Further, working with partners in the European Association of National Metrology Institutes (EURAMET) we are developing cross platform methods for the label-free localisation of antimicrobial agents and bacterial components within the biofilm. These platforms include 3D OrbiSIMS (secondary ion mass spectrometry) and Raman spectroscopy. Here we demonstrate the localisation of key biofilm components such as Pseudomonas Quinolone Signal (PQS) molecules in a 3D chemical map of the biofilm.
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- Missing Microbes and the Hygiene Hypothesis: New Challenges and Perspectives
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Diversity of eukaryotic gut microbiota of northern Thai populations
Gut eukaryome refers to the collection of fungi and protists in the gut. Until recently, all eukaryotic gut microbes were considered parasites and subject to elimination. Though this is true in some instances, critical evaluation of the literature reveals that most microbial eukaryotes are harmless often colonizing the human gut for long periods of time. Evidence is accumulating that the eukaryome plays important ecological roles in gut communities, as well as, in health and disease of the host. Nonetheless, systematic examination aiming to obtain baseline information of their prevalence and diversity in human populations is lagging. To address this knowledge gap, we collected fecal samples from a population of adults residing in north Thailand (n=211), who showed no gastrointestinal (GI) symptoms and had no history of GI diseases. We then examined the prevalence and diversity of two commonly found eukaryotic genera: the stramenopile Blastocystis (Blastocystis spp.) and the yeast Candida (Candida tropicalis and Candida albicans). Twenty three percent of individuals were positive for Blastocystis. Their sequences grouped in six of the nine clades that colonize humans. Twelve percent of the study population was positive for Candida, 4 % for C. albicans and 8 % for C. tropicalis, while concurrent colonization was also noted in some individuals. Eukaryotic bacterial interactions, as well as, interplay with diet and body mass index are also discussed. This is the first study providing data on the eukaryome of Thai populations and evidence that microeukaryotes traditionally considered as pathogenic asymptomatically colonize the gut of healthy humans.
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Assessment of novel disinfection technologies, and bacterial contamination in the healthcare setting
More LessWorldwide, hundreds of thousands of healthcare acquired infections (HAIs) are reported each year. Contamination of hospitals is a source of, and allows dissemination of HAIs. In healthcare settings one of the major vectors of contamination is healthcare workers’ uniforms. As surfaces become contaminated, bacteria can then be contacted by patients or staff who may indirectly spread bacteria to patients. Direct and indirect spread of bacteria could result in infection of patients and increased infection rates. Further consequences include increased levels of antibiotic use and costs. A pilot study was conducted at Antrim Area Hospital, Northern Health and Social Care Trust. 100 pre-shift and 100 post-shift healthcare workers’ uniforms were assessed for Staphylococcus aureus and Enterococcus spp. isolates. We found increased levels of antibiotic resistant S. aureus and Enterococcus spp. contamination on post-shift uniforms compared to zero to minimal contamination of pre-shift uniforms. A biobank of isolates was subsequently characterised for antibiotic sensitivity using European Union Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines – 51 % of S. aureus isolates were classed multi-drug resistant. Genomic diversity was assessed using Random Amplification of Polymorphic DNA (RAPD) – high levels of similarity was found amongst isolates. As one means of reducing uniform bioburden, we conducted analysis of a novel surface-active organisilane disinfectant named Goldshield (GS). GS was marketed as a long lasting antimicrobial prevent (re)contamination. GS technology displayed bactericidal, sporicidal and anti-biofilm properties in laboratory testing providing rationale for an intervention where GS could be incorporated into hospital laundry and assessed for potential use in infection control.
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