- Volume 4, Issue 5, 2022
Volume 4, Issue 5, 2022
- Abstracts from Annual Conference 2021
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- Poster Presentations
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Trans-cinnamaldehyde - geraniol mixture – antibacterial activity and haemotoxicity
More LessThe aim of this study was to test the antibacterial activity and haemotoxicity of (trans)-cinnamaldehyde and geraniol. Conducted analyzes showed that (trans)-cinnamaldehyde-geraniol mixture have antibacterial properties against S. aureus ATCC6538, E. coli ATCC8739 and P. aeruginosa KKP991. MIC and MBC of the mixture was determined for each strain. For S. aureus MIC=0.065 mg/ml, MBC=1.25 mg/ml, for E. coli MIC= 0.5 mg/ml, MBC=1 mg/ml and for P. aeruginosa MIC=0.5 mg/ml, MBC=1.25 mg/ml. Also the blood compatibility test was done, and it showed that MIC for S. aureus (0.025 mg/ml) is non-haemotoxic, but other MICs and all MBCs, unfortunately are.
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A potential new paradigm of denitrification in Neisseria gonorrhoeae
More LessMetals are bacterial nutrients. Upon infection by microorganisms, the animal host innate immune system typically reduces the availability of metals. In response, bacterial pathogens can activate pathways for metal uptake to avoid metal starvation. This competition for metals at the host-pathogen interface is termed “nutritional immunity”.
We are interested in how the obligate human pathogen Neisseria gonorrhoeae acquires nutrient copper (Cu). This Gram-negative bacterium expresses several respiratory cuproenzymes that are required for growth and metabolism in both aerobic and anaerobic conditions. These cuproenzymes include the putative nitrous oxide reductase (NosZ). NosZ contains 12 Cu atoms per functional dimer and it catalyses the reduction of nitrous oxide (N2O) to dinitrogen (N2). This reduction is an intermediate step in the denitrification pathway, in which nitrite (NO2-) is used as the terminal electron acceptor for respiration instead of O2. In this project, we will determine whether NosZ is expressed as a functional enzyme in N. gonorrhoeae. We will then examine how this enzyme acquires nutrient Cu and subsequently assembles its active site.
Given the rise in antibiotic resistance and the worldwide recognition of multidrug-resistant N. gonorrhoeae as a major threat to public health, we hope that a fundamental understanding of the physiology and metabolism of this organism will yield new strategies for anti-infectives, for instance by manipulating Cu availability at the site of infection.
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Phenotypic whole-cell screening identifies a protective carbohydrate epitope on Klebsiella pneumoniae
More LessThe increasing occurrence of recalcitrant multi-drug resistant (MDR) Klebsiella pneumoniae infections coupled with a diminishing pipeline of new antibioticswarrants the investigation of alternative antimicrobial therapies. We employed a target-agnostic phage display approach using live K. pneumoniaebacteria with the aim of isolating therapeutic monoclonal antibodies (mAbs) targetingconserved epitopes among clinically relevant strains. mAb targets were explored using ELISA and biolayer interferometry, and a high-throughput opsonophagocytic killing assay was developed to determine functional activity. Fluorescence-activated cell sorting was used to screen a global panel of clinical isolates, and high-content imaging further explored binding and functional activity. One mAb was tested in vivousing a lethal murine model of pneumonia. mAbs binding to carbohydrate epitopes were isolated in phage display selections enriched on wild-type and capsule-deficient strains. mAbs binding O1 lipopolysaccharide (LPS) and cross-binding O1/O2 LPS were identified. mAbs were shown to promote opsonophagocytic killing by human monocyte-derived macrophages, and clearance of macrophage-associated bacteria. One mAb, named B39, protected mice against MDR O1 and O2 strains when dosed therapeutically in a murine pneumonia model. Binding to a panel of O1 and O2 clinical isolates suggests B39 binds to both d-galactan-I and d-galactan-II of the LPS. With the rise of antimicrobial resistance among enteric pathogens, the discovery of a novel therapeutic mAb targeting the most prevalent K. pneumoniaeserotypes demonstrates a significant advancement in the field, and showcases the potential of alternative antimicrobial therapies for the treatment of MDR infections.
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Phenotypic diversity of a clonal Ralstonia solanacearum pathogen lineage is explained by accessory genome variation
More LessRalstonia solanacearumis aplant pathogenic gram-negative bacterium capable of infecting several economically important crops such as potato and tomato. It can also persist in environmental reservoirs including soils, rivers and in asymptomatic wild hosts, causing disease outbreaks during pathogen spillover events when crossing agroecological interface. In the UK, R. solanacearum outbreaks originate from Solanum dulcamarawild hosts (woody nightshade) and river networks. To what extent selection in these natural environments drive R. solanacearumsurvival and life history evolution including virulence is unknown. To study this, we focused on a largely clonal R. solanacearum lineage inhabiting river networks across the UK consisting of a collection of 182 isolates spanning 30 years since the first outbreak in 1992. We first characterised strains phenotypically regarding 32 traits including resource catabolism, virulence and abiotic stress tolerance and then used microbial GWAS techniques to identify links between phenotypic traits and the presence of specific accessory genes. We found that isolates can be clustered into three phenotypic groups, which differed clearly regarding their resource specialism and stress tolerance. No effect of isolation location was found. However, isolates became more variable phenotypically along with time. While only few SNPs were found to vary among all isolates, the presence and absence of certain accessory genes, such asS-layer family protein,could be associated with phenotypic variation in terms ofsurvival in stressful environments. Together, our findings suggest that a low number of accessory genes can cause high phenotypic variability within highly clonal bacterial lineage.
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4000 metres closer to novel antibiotics: a bioprospecting tale of three Streptomyces strains from the Andes
More LessBioprospecting of underexplored environments and microbiomes remains one of the core strategies for drug discovery. Páramos, a high-altitude ecosystem and evolutionary hotspot in the northern Andes of South America, harbour microbial diversity yet to be studied for its potential for antibiotic production. In this project, three strains (CG885, CG893 and CG926) isolated from a páramo in Colombia were explored using phylogenetic and genome mining tools to uncover their potential for novel antimicrobials. Taxonomic characterisation of three isolates suggested strains CG885 and CG893 can be classified as Streptomyces pratensis while strain CG926 is likely to be a new Streptomyces species. All three strains showed the biosynthetic coding capacity characteristic of streptomycetes and a diverse repertoire of biosynthetic gene cluster types likely to encode for novel specialised metabolites. One cluster in the strain CG926, for instance, is predicted to encode for a new halogenated compound containing the unusual nonproteinogenic amino acid piperazic acid with no known analogue. Integration of these results with phenotypic and metabolomic data will enable the assessment of these molecules and their antimicrobial activity. Overall, these results demonstrated that these three strains from an underexplored environment harbour the potential to become producers of novel antibiotics.
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Whole genome sequencing and comparative analysis of a novel Chlamydia psittaci strain
BackgroundChlamydia abortus and Chlamydia psittaci are important pathogens of livestock and avian species, respectively. While C. abortus is recognized as descended from C. psittaci species, there is emerging evidence of strains that are intermediary, including C. psittaci strain 84/2334 that was isolated from a parrot.
MethodsStrain 84/2334 was analysed by multi-locus sequence typing of seven housekeeping genes and by analysis of five species-discriminant proteins. The strain was de novo sequenced for comparison with representative strains from chlamydial species. 16S rRNA and whole genome phylogenetic analyses, network and recombination analyses, and analyses of key virulence-associated genes were conducted to explore its evolutionary relationship with both C. psittaci and C. abortus.
ResultsTyping, 16S rRNA and whole genome phylogenetic analyses, as well as network and recombination analyses showed that strain 84/2334 clusters closely with C. abortus. Analyses also suggested a closer evolutionary relationship with classical C. abortus strains, than to two other avian C. abortus or C. psittaci strains. Analyses of virulence-associated genes that exhibit greatest diversity within chlamydial species, reveal greater diversity than present in sequenced C. abortus genomes. The strain also possesses an extrachromosomal plasmid, as found in C. psittaci species but absent from all sequenced classical C. abortus strains.
ConclusionThe results are consistent with 84/2334 being a C. abortus ancestral strain, suggesting that it should be reclassified as C. abortus. The identification of a C. abortus strain bearing an extra-chromosomal plasmid has implications for plasmid-based transformation studies providing a potential route for future vaccine development.
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An interdisciplinary approach to reveal the dynamics of generalized transduction of antimicrobial resistance genes
More LessBackgroundAntimicrobial resistance (AMR) genes can spread between bacteria by “generalized transduction”, where phages act as vectors to transfer them. However, our knowledge of the dynamics of transduction and how to best represent them is limited. We aimed to fill this gap through an interdisciplinary approach, generating microbiological data and using mathematical models to clarify the underlying transduction dynamics.
MethodsWe co-cultured two library strains of Methicillin-resistantStaphylococcus aureus, each harbouring a resistance gene for a different antibiotic, with 80α generalized transducing phage. We recorded numbers of bacteria and phages at multiple time-points over 24h, using the presence of bacteria resistant to both antibiotics as evidence that transduction occurred. We developed and compared mathematical models of transduction based on how well they fit to the lab data.
ResultsAfter a growth phase of 8h, bacteria and phage surprisingly coexisted at a stable equilibrium in our culture, the level of which was dependent on the initial concentration of phage. The rate of transducing phage generation was approximately 10-6per lytic phage, sufficient to consistently generate double resistant bacteria, detectable after only 7h. Dynamics of transduction were best captured by a mathematical model in which the rate of phage infection slows as the bacteria population approaches carrying capacity.
ConclusionsThe novel data and models generated provide valuable insights into the dynamics of transduction of AMR. This interdisciplinary framework could be extended to other bacterial species, and is the first step towards evaluating the impact of transduction on the overall public health consequences of AMR.
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Evaluation of a monoglyceride for antimicrobial activity against Gram-positive and -negative bacteria
More LessBackgroundThe Gram-positive bacterium Staphylococcus aureus, and Gram-negative Neisseria gonorrhoeae and Pseudomonas aeruginosa bacteria, have developed multi-drug resistance to currently recommended antibiotic treatment resulting in worse clinical outcomes and significant economic burden. N. gonorrhoeae causes the sexually transmitted infection gonorrhoea and S. aureus and P. aeruginosa causes nosocomial infections including a plethora of infections that affect the skin, lungs, heart, eyes, ears, bones, and blood. Novel therapies are desperately needed to replenish the clinical pipeline as current antibiotic treatments fail.
MethodsMonolaurin was investigated against strains of N. gonorrhoeae, S. aureus and P. aeruginosa. The minimum inhibitory concentration of monolaurin was determined by agar diffusion assay for N. gonorrhoeae and broth microdilution assay for S. aureus and P. aeruginosa. Bactericidal activity of monolaurin was assessed in log reduction assay and the time - kill activity of various concentrations of the monoglyceride was evaluated over 5 hours. At least 4 log10 killing activity was considered bactericidal.
ResultMonolaurin inhibited growth ofS. aureus, N. gonorrhoeae, and P. aeruginosa at a range of 0.1mM - 3.13mM concentrations. At 1.5mM concentration, monolaurin killed S. aureus and N. gonorrhoeae at 180 minutes and 60 minutes, respectively. Monolaurin at 3mM concentration rapidly killed N. gonorrhoeae within 2 minutes while 25mM concentration killed P. aeruginosa within 60 minutes.
ConclusionThe evidence suggests that monolaurin actively killed Gram-positive and remarkably Gram-negative bacteria. This monoglyceride can potentially be developed as an antimicrobial therapy against these bacterial infections and may prove useful when coinfection with P. aeruginosa and S. aureus are present.
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Assessing the interspecies relationship betweenPseudomonas aeruginosa and Staphylococcus aureus in mixed biofilms grown in alginate beads and collagen scaffolds
More LessPolymicrobial biofilms in chronic infected wounds harbour different bacterial species that interact with each other, competing or co-operating to survive. The two most common pathogens co-isolated from chronic wound biofilms are Pseudomonas aeruginosa and Staphylococcus aureus. Evidence from in vitrobiofilms models have shown these two bacteria interactand data suggests that P. aeruginosa inhibits the growth of S. aureusin mixed biofilms. This study aimed to assess the growth of these two species in a complex polymicrobial biofilm in a 3D matrix comprised of either alginate (1.5% w/v) or a collagen scaffold.
Using a five-species biofilm (S. aureus, P. aeruginosa, Citrobacter freundii, Enterococcus faecalisand Escherichia coli), with all bacteria inoculated at time zero, it was consistently observed that P. aeruginosa was not recoverable over a 72h period, with sampling every 24h. However, P. aeruginosagrew well if it was added to a pre-formed four-species biofilm. Further, P. aeruginosa was seen to inhibit the growth of S. aureus after 24h subsequent co-culture in the pre-formed biofilm, which resulted in the emergence of small colony variants of S. aureus. Interestingly when P. aeruginosawas co-inoculated in a four species biofilm that did not contain S. aureus,its growth was not inhibited, suggesting a competitive interaction between these two bacteria during establishment of the early biofilm. These data were consistent in alginate beads and collagen scaffolds. In a chronic wound P. aeruginosais regarded as a late coloniser and the phenomena observed in this study might be reflective of this.
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Examining Phosphate Physiology in Coccolithophore Life Cycle Stages
More LessDespite the significance of coccolithophores for biogeochemical cycling, much of their ecology remains poorly understood. In particular, their elusive haplo-diplontic life cyclehas been given little attention in the literature. Yet, it significantly impacts the extent of coccolithophore calcification, their vertical and horizontal distribution in the ocean, and potentially their global success.
The aim of this project is to establish physiological differences between the life cycle stages of the ecologically important coccolithophores Coccolithus braarudiiandCalcidiscus leptoporus in response to changes in the essential macronutrient phosphorus (P). Haploid coccolithophores are more commonly found in P-limited environments, raising the question which underlying mechanisms cause this variable distribution between life stages.
An initial investigation of growth, photophysiology, calcification, and storage of polyphosphate under P-limited conditions will determine which parts of coccolithophore physiology are heavily impacted by P-limitation. An in silicoanalysis of P acquisition and transport genes in coccolithophores and other closely related eukaryotes will highlight differences in P physiology among these groups. Comparing proteins expressed in haploid and diploid coccolithophores under P-limitation will then be used to reveal whether P acquisition strategies differ between the two life stages.
These results will further shed light on potential adaptations of haploid and diploid coccolithophores to different ecological niches. A profound understanding of coccolithophore physiology is vital to revealing their evolutionary success and their impact on ocean biogeochemistry and ecology.
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Regulatory mutants of Serratia with modulated antibiotic production due to intergenic transposon insertions
More LessAntibiotic resistance is an increasing problem, exacerbated by global dissemination of drug resistance genes under selection pressure. Moreover, the rate of new antibiotic discovery declined over previous decades and so there is a growing need for new antibiotic discovery and a deeper appreciation of the various genetic and physiological factors that influence antibiotic biosynthesis.
The enterobacterium Serratia sp. ATCC 39006 (Serratia) is a useful model for studies on the biosynthesis and regulation of bioactive secondary metabolites, including two antibiotics - a carbapenem and prodigiosin. Both compounds are tightly regulated in response to various physiological and environmental signals, including quorum sensing.
We have identified novel regulators of antibiotic production after random transposon mutagenesis. Multiple regulatory mutations mapped to a small locus defining an intergenic region (IGR). These IGR insertion mutants display elevated production of both the carbapenem and prodigiosin antibiotics and they also exhibit reduced motility, confirming pleiotropic impacts. Exploiting phage transduction, we constructed double mutants with 15 known Serratia regulators and showed that the IGR mutant phenotype was epistatic over several of these. Further analysis suggested the presence of a putative gene encoded within the IGR locus, which may play an impactful role in the intricate regulatory network. Functional characterisation of the IGR region and its physiological impacts in both the modulation of antibiotic production and in wider pleiotropy were dissected. Mechanistic understanding of the regulatory mechanisms involved may prove exploitable in enhancing controlled antibiotic hyperproduction.
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SARS-CoV-2 Spike has broad tropism for mammalian ACE2 proteins yet exhibits a distinct pattern of receptor usage when compared to other β-coronavirus Spike proteins
Carina Conceicao*, Nazia Thakur*, Stacey Human, James T Kelly, Leanne Logan, Dagmara Bialy, Sushant Bhat, Phoebe Stevenson-Leggett, Adrian K Zagrajek, Philippa Hollinghurst, Michal Varga, Christina Tsirigoti, Matthew Tully, Chris Chiu, Katy Moffat, Adrian Paul Silesian, John A Hammond, Helena J Maier, Erica Bickerton, Holly Shelton, Isabelle Dietrich, Stephen C Graham and Dalan BaileyThe Coronavirus Disease 2019 (COVID-19) pandemic, caused by SARS Coronavirus 2 (SARS-CoV-2), continues to cause significant mortality in human populations worldwide. SARS-CoV-2 has high sequence similarity to SARS-CoV and other related coronaviruses circulating in bats. It is still unclear whether transmission occurred directly from bats to humans, or through an intermediate host, bringing into question the broader host range of SARS-CoV-2. Using a combination of low biocontainment entry assays as well as live virus, we explored the receptor usage of SARS-CoV-2 using angiotensin-converting enzyme 2 (ACE2) receptors from 22 different species. We demonstrated that in addition to human ACE2, the Spike of SARS-CoV-2 has broad tropism for other mammalian ACE2s, including dog, cat and cattle. However, comparison of SARS-CoV-2 receptor usage to the related SARS-CoV and bat coronavirus, RaTG13, identified distinct patterns of receptor usage, with the two human viruses being more closely aligned. Finally, using bioinformatics, structure analysis and targeted mutagenesis, we identified key residues at the Spike-ACE2 interface which may have played a pivotal role in the emergence of SARS-CoV-2 in humans, some of which are also mutated in newly circulating variants of the virus. To summarise, the broad tropism of SARS-CoV-2 at the point of viral entry identifies the potential risk of infection of a wide range of companion animals, livestock and wildlife.
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Identification of Novel Combinatorial Drug Targets in Mycobacterium tuberculosis
More LessTuberculosis caused by the Mycobacterium tuberculosis complex (MTBC) remains one of the most important infectious diseases of mankind. Isoniazid (INH) and rifampicin are the main first line drugs used in multidrug treatment of TB but phenotypic tolerance and the development of resistance against these compounds and other TB drugs is a serious and increasing problem. The overall aim of this project is to utilise transposon sequencing (Tnseq) genetic screens of MTBC to identify novel drug targets that function maximally when in combination with INH or rifampicin or increase the sensitivity to these drugs. Drugs directed to these targets could be developed as part of new multidrug therapies. INH is a prodrug which requires activation by the mycobacterial catalase/peroxidase, KatG to forman isonicotinoyl acyl radical, which binds to NAD+/NADH and inhibits enoyl-[acyl-carrier-protein] reductase (InhA)-dependent synthesis of cell wall mycolic acid. A Tnseq screen of BCG in a sub-MIC concentration of INH identified genes whose disruption increased apparent sensitivity to INH. A subset of these genes were functionally related to oxidative stress and were selected for further investigation of individualwild-type strains, mutants and complemented mutants. KatG activity was measured with a catalase assay to test the hypothesis that absence of the oxidative stress gene leads in the production of more KatG which allows for increased activation of the drug INH and increased sensitivity to INH. In a second Tnseq screen, mutants were identified with enhanced sensitivity to rifampicin. The significance of these genes will be investigated further and discussed.
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Evaluation of Drug Activity Against Staphylococcal Biofilms – Settings of Conditions Leading to Reproducible and Robust Biofilm Formation in vitro
More LessStaphylococcus aureus(SA)andepidermidis(SE) are the most common pathogens of the genus Staphylococcus, causing biofilm-associated infections. Bacteria in biofilms are difficult to eradicate due to their resistance and serve as a reservoir for recurring persistent infections. A variety of protocols for in vitro drug activity testing against biofilms has been introduced. However, there are often fundamental differences. In our pilot study, we developed optimal conditions for staphylococcal biofilm formation on plastic pegs in order to set a methodology for an evaluation of the antibiofilm activity of candidate molecules. The convenience of the plastic pegs lies in their removability from the lid for easy access to multiple equivalent biofilms, and in possibility of in situ detection and quantification by confocal laser microscopy. For the purpose of enhancement in staphylococcal biofilm formation, the impact of peg surface modification with 3 different coating materials was studied as well. An increase of biofilm biomass was evaluated by crystal violet staining method. The basic precondition for obtaining relevant and reproducible data regarding antibiofilm activity is the formation of robust biofilms with typical attributes such as the presence of a biofilm matrix. In our study, in vitro conditions revealed that we fully met the preconditions for the SA and methicillin-resistant SA strains. In conclusion, we demonstrated statistically significant enhancement of biofilm formation in all studied staphylococcal strains, including either strong biofilm producer phenotype (SA, methicillin-resistant SA) and weak biofilm producer phenotype (SE).
Supported by the SVV Project No. 260549 and by the Czech Science Foundation project No. 20-19638Y.
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Immunoproteomic analysis of surface proteins in bovine-adapted lineages of Staphylococcus aureus
More LessIn Ireland, Staphylococcus aureus is the most frequent cause of bovine intramammary infection (IMI) with the bovine-adapted lineages CC151 and CC97 most commonly found. While bovine mastitis vaccines are available that target S. aureus, this pathogen is still a significant source of infection in dairy cows. Therefore, there’s a clear need for a more effective vaccine against S. aureus. However, S. aureus is considered a clonal organism, therefore identifying new potential protein targets common to all major lineages is an important step for vaccine design.
Two S. aureus strains, one belonging to ST151 and another to ST3170 (CC97), were used to infect two groups of dairy cows. Antibodies raised by individual cows were used to identify immunodominantsurface proteins for each strain. One-dimensional serum blotting determined that the antibody response to S. aureus infection was largely strain specific, and to a lesser extent, animal specific.
Two-dimensional serum blotting followed by mass spectrometry of immunoreactive spots was used to identify potential vaccine candidates that were immunodominant for both strains. These blots showed that proteins to which an antibody response was generated in the ST151 infected cows were generally different to those generated in the ST3170 infected cows. However, mass spectrometry also identified immunoreactive proteins common to both strains including Clumping factor B and Iron-regulated surface determinant protein A. Common immunoreactive secreted proteins are also currently being identified.
This study will identify immunodominant proteins expressed by the bovine-adapted strains ST3170 and ST151 that could potentially be used as candidates in vaccine research.
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Characterisation of the Rh50 protein from the ammonia-oxidising bacterium Nitrosomonas europaea
The transport of ammonium through the cell membranes, an essential process in all kingdoms of life, is accomplished by the ubiquitous Amt/Mep/Rh superfamily of proteins. The functional context of Amt/Mep and Rh transporters is diverse: bacteria, fungi, and plants use Amt/Mep proteins to scavenge ammonium for biosynthetic assimilation, whereas mammals use the Rh proteins for ammonium detoxification in erythrocytes, kidney, and liver tissues. While RH50 genes are widespread in eukaryotes they are present in some prokaryotes: an example is a chemolithoautotroph Nitrosomonas europaeawhich gains all its energy from the oxidation of ammonia to nitrate.
While Amt/Mep/Rh proteins have divergent physiological functions, they are structurally very similar, which raises the important question about the universality of the transport mechanism. We have recently proposed an elegant new model for the mechanism of electrogenic ammonium transport in bacteria Amt protein: after deprotonation of NH4+ at the periplasmic side of the transporter, a previously undiscovered polar conduction route enables H+ transfer into the cytoplasm. A parallel pathway, lined by hydrophobic groups within the protein core, facilitates the simultaneous transfer of uncharged NH3. In this context, we propose to elucidate at the molecular level the mechanism of ammonium translocation through rhesus protein from Nitrosomonas europaeaand establish whether there is a universal mechanism for biological ammonium transport. Beyond the elucidation of a central biological process, this work has important medical implications, as some Rh mutations have been associated with human pathologies. We propose to demonstrate how specific Rh mutations affect the activity of the protein to establish the relationship between Rh malfunction and the associated diseases.
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Use of long read sequencing to characterise the genomic architecture of mobile genetic elements encoding blaCTX-M-15 in Escherichia coli causing travellers’ diarrhoea
More LessIncreasing levels of antimicrobial resistance (AMR) have been documented in Escherichia colicausing travellers’ diarrhoea, particularly to the third-generation cephalosporins. Diarrhoeagenic E. coli (DEC) can act as a reservoir for the exchange of AMR genes between bacteria residing in the human gut, enabling them to survive and flourish through the selective pressures of antibiotic treatments.
Using Oxford Nanopore Technology (ONT), we sequenced eight different sequence types (ST) belonging to five different pathotypes of extended-beta-lactamase-producing DEC harbouring blaCTX-M-15from four patients recently returned to the UK from Pakistan. The aim of the study was to determine whether blaCTX-M-15 was chromosome or plasmid-encoded to better understand the mechanisms of onward transmission of AMR determinants.
In Patient A, blaCTX-M-15was plasmid-encoded in both DEC isolates (ST504/ST3032), whereas in Patient B blaCTX-M-15was located on the chromosome in both DEC isolates (ST227/1283). Patients C and D both had one isolate where blaCTX-M-15 was located on the plasmid and one chromosomally encoded (ST443/182 and ST38/99, respectively). The two plasmids associated with Patient A were different although one exhibited high similarity to the plasmid from Patient C. In the four isolates where blaCTX-M-15 was chromosomally encoded, the site of insertion and the characteristics of the inserted plasmid segment differed.
Analysis of long-read sequencing data enables us to characterise the genomic architecture of mobile genetic elements encoding AMR determinants. These data may contribute to a better understanding of persistence and onward transmission of AMR determinants in MDR E. coli causing gastrointestinal and extra-intestinal infections.
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Bioinformatic exploration of trimethylamine N-oxide metabolism in human gut bacteria
More LessTrimethylamine N-oxide (TMAO) is a microbial metabolite that has been shown to have protective effects on the blood–brain barrier, while elevated serum levels of TMAO and its precursors have been linked to cardiometabolic diseases in Western populations. Previous work examined the prevalence of TorA to determine which groups of bacteria were responsible for the metabolism of TMAO in the human gut. This study examined 6 TMAO metabolism pathways to provide a more in-depth analysis of bacterial TMAO metabolism. These results were then filtered for hits with >90% coverage and >70% identity.
Results showed that Tor proteins were largely limited to members of the Enterobacteriaceae, mostly appearing in Escherichia coli and Citrobacter spp. >1% of 9898 Klebsiella spp. genomes examined encode any Tor proteins, despite previous work highlighting Klebsiella spp. as one of the prevalent genera encoding TorA. Dms proteins were much more prevalent than TorA in other genera of bacteria, along with MsrP and BisC. 118 of the HGRGs were found to encode for at least 1 TMAO metabolism protein.
Overall, this work highlights the need for more comprehensive methods to be used to examine large genomic and metagenomic datasets and the need for in vitro work to be done alongside in silico analyses to improve functional annotations and our understanding of the roles of gut bacteria.
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A Campylobacter integrative conjugative element with a CRISPR-Cas9 system targeting competing plasmids: a history of plasmid warfare?
More LessMicrobial genomes are highly adaptable, with mobile genetic elements such as integrative conjugative elements (ICE) mediating the dissemination of new genetic information throughout bacterial populations. This is countered by defence mechanism such as CRISPR-Cas systems, which limit invading mobile elements by targetting specific sequences on these elements. Here we have studied the distribution the pVir, pTet and PCC42 plasmids and a new 70-129 kb ICE (CampyICE1) in the foodborne microbial pathogens Campylobacter jejuni and Campylobacter coli. CampyICE1 contains a degenerated Type II-C CRISPR system consisting of a sole Cas9 protein, which is distinct from the previously described Cas9 proteins from C. jejuni andC. coli. CampyICE1 is highly conserved in structure and gene order, containing blocks of genes predicted to be involved in recombination, regulation and conjugation. CampyICE1 was detected in 134/5,829 (2.3%) C. jejunigenomes and 92/1,347 (6.8%) C. coligenomes. Similar ICE were detected in a number ofnon-jejuni/coli Campylobacter species, which lacked a CRISPR-Cas system. Finally, CampyICE1 contained 3 separate short CRISPR spacer arrays, and a total of 124 unique spacers were identified, of which 67 are predicted to target the Campylobacterplasmids pVir, pTet, and pCC42, and 12 predicted to target other Campylobacter plasmids (63.7%). The presence of a functional CampyICE1 Cas9 protein and matching anti-plasmid spacers was associated with the absence of these plasmids (186/214 genomes), implicating that the CampyICE1-encoded CRISPR-Cas has contributed the exclusion of competing plasmids. Hence the CampyICE1 CRISPR-Cas system may be a part of ongoing plasmid warfare in Campylobacter spp.
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Evaluation of essential oils efficacy on antibiotic-resistant Salmonella strains
BackgroundThe emergence of multidrug resistant (MDR) pathogens has drawn attention to natural antimicrobial compounds, such as essential oils (EOs)
MethodsThe aim of the present study was to investigate the inhibitory activity of three EOs against 15 Salmonella strains resistant to 16 commonly used antimicrobials. EOs of O. vulgare, T. serpyllum, and T. vulgariswere tested in triplicate for 15 bacterial strain by Kirby-Bauer disk diffusion method and broth microdilution method to determine the minimal inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC), according to the Clinical and Laboratory Standards Institute guidelines with minor modifications.
ResultsO. vulgare and T. serpyllum showed high antimicrobial activity compared to T. vulgaris in all tested antibiotic-resistant strains (P-value <0.01). The mean diameter of the bacterial growth inhibition zone was 18.7 mm for O. vulgaris, 19.2 mm for T. serpyllum, and 14.2 mm for T. vulgaris. The MIC and MBC mean values of O. vulgare were respectively 0.037% and 0.058%, for T. serpyllum 0.036% and 0.041%, for T. vulgaris 0.13% and 0.15%. All strains resulted resistant totrimethoprim-sulamethoxazoleand 14 strains resulted resistant to tetracycline, considered a highly important antimicrobial. Among Salmonella serovars tested the monophasic S. Typhimurium has exhibited the highest antimicrobial resistance to synthetic molecules while they were sensitive to EOs.
ConclusionsThe EOs of O. vulgare and T. serpyllum, which contain carvacrol as the main constituent, showed the greatest antimicrobial effect against MDR Salmonella strains.
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Functional responses of neutrophils to Fusobacterium nucleatum subspecies
More LessIntroductionFusobacterium nucleatum is an opportunistic oral pathogen with five subspecies: animalis, fusiforme, nucleatum, polymorphumandvincentii. These play an important role in biofilms associated with chronic gum disease (periodontitis). Neutrophils form the first line of defence against these and other oral pathogens, employing a multitude of anti-microbial strategies, which, alongside bacterial killing, also cause collateral host-tissue damage. The immunogenic properties of F. nucleatum subspecies are poorly understood. The purpose of this study was to investigate specific neutrophil responses to all F. nucleatum subspecies.
MethodsAllF. nucleatumsubspecieswere grown planktonically overnight and inactivated using 10% neutral buffered formalin. Peripheral blood neutrophils (PBN) and neutrophil-like cells (differentiated from HL-60 cells) were challenged with inactivated bacteria (MOI 100) and production of reactive oxygen species (ROS) was quantified by Luminol/Iso-luminol enhanced chemiluminescence assays. Release of neutrophil extracellular traps (NETs) was quantified fluorescently using Sytox-green. Additionally, phagocytosis of fluorescently labelled F. nucleatum subspecies was analysed in both cell groups by flow cytometry.
ResultsIn addition to differences in ROS generation by PBN and neutrophil-like cells in response to individual F. nucleatum subspecies, variations in the amount of extracellular DNA extruded during NETosis was evident. Moreover, distinct levels of phagocytic activity of both cell types will be shown.
ConclusionF. nucleatum pathogenicity has been reported in oral as well as systemic diseases (colorectal cancer, cardiovascular disease). Understanding subspecies-specific effects of F. nucleatum on neutrophils will enhance our knowledge of the interactions between this bacterium and the host immune response and may help to identify new therapeutic targets.
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Perfluorooctanoic acid (PFOA) sensitises E. coli to acid stress
More LessPer-and Polyfluoroalkyl Substances (PFAS), which includes PFOS and PFOA, are recognized as the most important class of emerging contaminants due to their widespread presence in wildlife and humans, their environmental persistence, bioaccumulative potential, and toxicity. Low environmental pH conditions affect the state and physicochemical properties of PFAS, which in turn impacts on their toxicity. Sparse information is available regarding the effect of these compounds on microorganisms, and the possible associated knock-on effect on ecosystem services. The purpose of this study is to investigate the ability of E. coli to tolerate stress in the presence of PFAS compounds. To this end, we carried out phenotypic comparisons of E. coli exposed to a series of environmental conditions in the presence and absence of PFOA and PFOS. Quite remarkably, E. coli growth was not affected by the presence of PFOA and PFOS up to 500 mg/L. The survival of E. coli at pH3, however, decreased by more than three-fold when the medium was supplemented with PFOA. PFOA and PFOS were also found to decrease the growth rates of E. coli in minimal media in the presence of 0.75 and 1M NaCl. We are currently screening further stresses of environmental relevance with the aim to conduct molecular investigations to examine the mechanisms underpinning the effect of PFAS exposure on bacterial stress tolerance. This work will provide some insights into the impact of PFAS on microorganisms, which should shed new light on the assessment of the ecological effects of PFAS.
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Parallel phage resistance - virulence trade - offs during clinical phage therapy and in vitro
With rising antibiotic resistance, modern medicine needs new approaches for tackling bacterial infections. Phage therapy uses the viruses of pathogenic bacteria to clear the infection. Unlike antibiotics, phage can evolve if bacteria become resistant to maintain or even increase their infectivity (coevolution). While laboratory studies can give insight into complex bacteria-phage interactions, whether they act as a true representation of phage therapy in patients is unknown. Here, we compared phage therapy in a patient to that of in vitro experiments. The patient had been admitted with aPseudomonas aeruginosainfection and was successfully treated with a phage cocktail. Bacteria were isolated before and during phage therapy, allowing us to follow bacteria-phage coevolution in the patient while doing experiments on the same clones in vitro. In vivo and in vitro, bacteria rapidly evolved resistance with little or no evidence of bacteria - phage coevolution. Although resistance mechanisms differed, parallel resistance - virulence trade-offs were found in vivo and in vitro. Therefore, phage resistance could increase treatment success and our results indicate to what extent bacteria - phage evolutionary dynamics can be predicted from in vitro experiments.
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Potentials of Breast Milk Antibodies as a Therapeutic for COVID-19
More LessSince the explosion of pneumonia-like symptoms in the province of Hubei, Wuhan in December 2019 over 200 countries have gotten a share of this disease caused by the novel coronavirus disease 2019 (COVID-19). Till date, there exists no modest therapeutic agent to combat this virus. Antibodies in the breast milk of convalescent lactating mothers could be the silver bullet that would deflate the ballooning morbidity and mortality rate caused by the COVID-19 pandemic. Coupled with the fact that breast milk contains a very high amount of Immunoglobulin A which has a desirable higher in vivo stability than other antibodies. This novel approach of purifying antibodies from the breast milk of convalescent lactating mothers is a non-invasive technique relative to the conventional method of using convalescent plasma which had proven effective for generating passive immunity against the seasonal flu. Purified Immunoglobulins would be obtained by adding an equal volume of ammonium sulphate solution to the breast milk sample to induce a lyotropic effect and thereafter passing the partially purified solution through a column chromatographic system impregnated with a Jaclin –lectin, Protein A, L and G. Neutralizing antibodies would by assayed using the Enzyme-Linked Immunosorbent Assay technique. Various statistical tools will be used to analyze a double-blinded randomized clinical trials to ascertain the efficacy of the purified antibodies as it is expected to have clear cut effectiveness over the control and potentially become the invaluable in containing the pandemic.
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Environment and the Evolutionary Trajectory of Horizontal Gene Transfer
More LessGene acquisition through horizontal gene transfer (HGT) may either exert a beneficial, neutral, or deleterious fitness effect on the recipient cell thereby determining the evolutionary fate of the newly transferred gene. The distribution of fitness effects (DFE) thus is a fundamental predictor of the outcome of an HGT event.
The environment plays a considerable role in altering the fitness of a horizontally transferred gene. We have studied the DFE of genes transferred from Salmonella enterica serovar Typhimurium to Escherichia coli in six environments, that potentially represent the conditions experienced by the two species. The data suggests high variability, with some genes becoming deleterious in one environment, while becoming neutral or even beneficial in another, suggesting that fluctuating environments may increase the likelihood of HGT.
The present study focuses on the DFE of genes transferred from Escherichia coli to macrophage-associated Salmonella Typhimurium strain 4/74 in four environments, that mimic the conditions inside a macrophage cell. In addition to the external environment, we are also looking at, how changes in the intrinsic environment of a cell, after an HGT event, could affect fitness. Functional similarity of the horizontally transferred gene to the endogenous copy can cause an imbalance due to increased protein dosage, thereby leading to a negative fitness effect. By comparing the growth rates of each ortholog gene with the ‘wild type’, in the four environments, we can elucidate when gene dosage acts as a barrier to HGT, helping us to understand the relationship of environmental quality and HGT, which is of evolutionary importance. We have identified 12 genes showing dosage dependent effects across the four environments, with some genes showing environment specificity, indicating an increased intolerance of the recipient to high protein dosage, making it a significant barrier to HGT.
A preprint is available on bioRxiv https://doi.org/10.1101/2022.04.01.486712
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Back to soil: Awakening the production of cryptic antibiotics in Streptomyces
More LessMost of the antibiotic biosynthetic gene clusters (BGCs) in Streptomyces are not expressed under laboratory conditions, however these clusters are maintained in the genomes, therefore indicating that they must play important roles in adaptation and survival within their ecological niches.
Understanding the global regulation patterns that affect transcription of antibiotic BGCs in soil conditions versus laboratory conditions allows us to identify novel routes for up- or down- regulation in order to trigger expression of these BGCs and therefore production of these antibiotics under controlled laboratory and fermentation conditions. Cryptic pathways represent an untapped resource in terms of new metabolites that could be very useful in the clinic if we can awaken their expression and production.
We are using RNA sequencing to analyse global transcriptional patterns of S. venezuelae and S. rimosus grown in sterile soil, non-sterile soil and standard laboratory solid medium, as well as using an unbiased metabolomics approach to identify compounds produced in soils. These results will enable the identification of global regulatory pathways that activate cryptic clusters in nature and lead to the production of these compounds in soil.
This will enable us to generate genetically modified strains engineered to increase the yield of both well characterised and cryptic clusters which encode potentially novel compounds under industrial fermentation conditions. It is worth noting that the transcriptional data may also help shed light on the complex regulation of the life cycle of these Streptomyces strains in an ecologically relevant environment.
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Investigating the microbiological risks associated with urban flooding in the UK
More LessOver the last 30 years, the frequency and occurrence of intense rainfall, and thus extreme hydrological events –flooding- has steadily increased. Drainage infrastructure in the UK was not designed for a changing climate, and many sewer systems in densely populated urban areas, are unable to cope. Sewage overflow and surface run off in urban areas can act as vectors for the dissemination of pathogens, known to cause disease among human populations. Most of the previous studies in this field have focused on using faecal indicators such as E.coli when assessing the public health risk of floodwater [1]. However, traditional indicators do not accurately reflect the true risk that urban flooding poses [2]. Little is understood in regards to the survivability and behaviour of pathogens in different urban settings, which are fundamental to determine potential risks to public health.
Previous investigations in UK waterlogged soils have shown a clear response of microbial communities to water table variation, temperature, and nutrient availability in soil profiles [3]. This research aims to investigate, using advanced molecular methods, the dynamics of pathogens (i.e. movement through soil and survival rates), and microbial interactions at the soil/water interface- collecting information from field work studies and laboratory-controlled experiments.
The outcomes from this research will inform future management strategies of flooded sites that will aid to protect public health.
References
[1] Yard et al., 2014. J. Environ. Sci. Health 49, 1236-1243.
[2] Hussain, 2019. Freshwater Microbiology 10, 393-420.
[3] Douterelo et al., 2009. Int. Biodeterior. Biodegradation 63, 6, 795-805.
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Sorghum beer production using Saccharomyces sp. isolated from palm wine as the pitching yeast and Garcinia kola as the bittering agent
Beer production in Nigeria is dependent on imported brewery yeasts and hops, which consequently increases the cost of beer production. The current study was conducted to determine the effect of using indigenous strains of Saccharomycessp. in place of brewer’s yeast, and Garcinia kola as the bittering agent on beer quality. Saccharomycesspecies strains were isolated from palm wine, andSaccharomycescerevisiae specie selected using nitrates and lysine assimilation test. IndigenousS. cerevisiaewas used for beer brewing at different pitching rates (1×107, 2×107, & 3×107) using brewer’s yeast as the control. The effect of various concentrations ofGarcinia kola(0.1 ml, 0.2 ml and 0.3 ml) on beer taste and quality was later determined; beer brewed with hops served as the control sample. The physicochemical analysis showed that the pH, specific gravity (SG) and free amino nitrogen (FAN) values of beer brewed with local S. cerevisiae strain were between 4.32 - 4.52, 0.962 - 1.008, and 81.80 - 263.64 g/L, respectively while the alcohol value was from 5.12 - 11.16 ABV. However, pitching with 1×107 cells gave the best product and was therefore used for further analysis. Beer samples from wort pitched with local yeasts and brewed withGarcinia kolagave good quality beer comparable to the standard product. pH values of the products were in the range of 3.18 - 4.41, SG (0.962 - 1.006), FAN (16.87 - 81.80g/l), bitterness (8.83 - 17.83 EBU) and alcohol (5.38 - 11.16 ABV).
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The role of staphylococcal bacteriocins in nasal competition
More LessBacteriocins are antimicrobial toxins produced by bacteria to defend and invade territories by killing unrelated strains and species. Understanding if bacteriocins shape natural populations is important for understanding the evolution of antimicrobial resistance and identifying novel antimicrobials for clinical use. Staphylococcus aureus is an opportunistic pathogen that asymptomatically colonises the nasal cavity of 1 in 3 healthy adults. S. aureus is known to produce many different bacteriocins, however we are yet to understand the extent to which they mediate the establishment of nasal populations. Here, we test the importance of bacteriocins in driving colonisation success, by screening S. aureus antimicrobial inhibition against otherS. aureus strains and three commensal species that commonly co-inhabit the nasal cavity. We use a longitudinally sampled collection of 173S. aureusnasal isolates from 14 participants over 90-months to track within-individual population changes over time. We found that 8% of allS. aureus isolates produced bacteriocins active against other species, but that between-strain bacteriocin inhibition inS. aureus was very rare, observed by only 0.5% of isolates. Therefore, while there is no evidence that intraspecific competition drives colonisation success in the nasal cavity, interspecific competition is more likely to influence strain and species dominance.
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Comparison of lentiviral and vesicular stomatitis virus core SARS-CoV-2 pseudotypes and generation of a stable cell line for use in antibody neutralisation assays
Betacoronavirus SARS-CoV-2, the causative agent of COVID19, is a single stranded positive sense RNA virus. Since its emergence there has been great efforts to identify correlates of protection,which is crucial for vaccine evaluation studies. However, handling SARS-CoV-2 requires BSL-3 containment facilities slowing research efforts. Pseudotype viruses (PV) are a safe alternative to authentic virus that can be handled at low containment. PVs are chimeric viruses containing the core of a virus where its genome has been completely or partially replaced by a reporter gene, displaying a correctly folded SARS-CoV-2 spike on its surface. We developed lentiviral and vesicular stomatitis virus (VSV) core PVs alongside a stable A549 cell line expressing receptor ACE2 and protease TMPRSS2 responsible for S protein priming, for use in neutralization assays. Lentiviral PVs were generated by transfection with plasmids encoding the spike, HIV-1 gag-pol and a luciferase reporter. For VSV PVs, producer cells pre-transfected with the spike were infected with recombinant VSV expressing luciferase,before harvesting. The stable A549 cell line was generated by sequential infection of VSV-G PVs bearing lentiviral vectors encoding ACE2 and TMPRSS2 genes followed by antibiotic selection, before being tested in neutralization assays. We compared lentiviral and VSV PV platforms using monoclonal antibodies and convalescent sera with our stable A549 cells or HEK293T cells pre-transfected with plasmids encoding ACE2 and TMPRSS2. Antibody titres showed equivalence however VSV had the advantage of a shorter incubation therefore enabling a higher throughput. PVs offer a robust platform for future seroepidemiology and vaccine evaluation studies.
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Characterization of a Multidrug-ResistantCitrobacter pasteurii isolate carrying blaKPC-2 from municipal wastewater
More LessA carbapenem-resistant strain, UL-CPE-01, was isolated from a domestic wastewater treatment plant and subjected to antibiotic susceptibility testing showing resistance to a wide range of antibiotics. Complete genome and bioinformatic analysis of UL-CPE-01 was performed to investigate the nature of its resistance determinants and the strain identified as Citrobacter pasteuriibased onANI score. Citrobacter spp. are part of the normal human and animal intestinal flora and the UL-CPE-01 Citrobacter pasteuriistrain detected here is closely related to Citrobacter freundii, which is an emerging opportunistic nosocomial pathogen. UL-CPE-01 showed both phenotypic and genotypic multiple resistance to aminoglycoside, β-lactams, sulfonamides, fluoroquinolone, rifampicin, phenicol and macrolide antibiotics. The UL-CPE-01 genome revealed 16 acquired antimicrobial resistance gene (ARGs) conferring resistance to eight different antibiotic groups: dfrA12 (trimethoprim); sul1 (sulfonamide); mph(A), mph(E) and msr(E) (macrolide); qnrB4 and aac(6′)-Ib-cr (fluoroquinolone); blaDHA-1, blaOXA-1, blaCMY-77,blaOXA-10, blaTEM-1B, blaKPC-2(β-lactams); arr-3 (rifampicin); catB3 and catA1 (chloramphenicol) and aac(6′)-Ib-cr, aadA2 and ant(2″)-Ia (aminoglycoside). The novel blaKPC-2 determinant was found to be associated with in a Tn4401-like element.
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Histopathological analysis of placental lesions caused by Chlamydia abortus 1B vaccine strain in vaccinated ewes
BackgroundChlamydia abortus is one of the most diagnosed causes of infectious abortion in small ruminants. Infections can be controlled using the live, attenuated C. abortus strain 1B vaccine, which has been associated with infection and abortion in animals. This study aimed to compare the severity and the distribution of lesions caused by this vaccine strain (vt) with those resulting from a wild-type (wt) infection.
MethodsTwo grossly affected and 1B-positive (by qPCR and RFLP analysis) placentas from a vaccinated sheep flock were analysed. Histopathological lesions and immunohistochemical labelling (IHC) were graded (increasing score from 0 to 5) according to their severity and distribution. Pathology in the vt infected placentas was compared with that in two wt infected placentas. Datasets generated for observed histological and pathological features were analysed using principal component analysis (PCA).
ResultsHistopathologically, the lesions in both vt and wt-placentas presented as typical multifocal necrosuppurative placentitis, associated with vasculitis, mural necrosis, and thrombosis. IHC for C. abortus revealed intense staining with a multifocal distribution in most cotyledons in both vt and wt placentas. Comparison of the pathological lesions between vt and wt by PCA revealed a similar distribution and severity, revealing a strong association with features such as necrosis and inflammatory infiltration between vt and wt placentas. A weaker association with IHC was observed.
ConclusionThis study shows that the C. abortus attenuated 1B vaccine strain presents no significant differences in severity and distribution of pathological lesions from those typically observed following a wt infection.
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Making virus taxonomy accessible
The International Committee on Taxonomy of Viruses (ICTV) is responsible for approving changes to the taxonomy of viruses. These changes arise from proposals made to the ICTV, usually by one of its 101 Study Groups, each of which is responsible for a particular virus family or families. The result of this international, voluntary effort is a searchable and structured list of current virus taxonomy that can be accessed on the ICTV website (www.ictv.global). The website also makes available the virus metadata resource (VMR), a spreadsheet with exemplar isolates and GenBank accession numbers for members of each species, as well as the ICTV Report which provides more detail about the taxonomy, structure and biology of each virus family. The online publication mode allows taxonomic changes and relevant research findings to be quickly reflected in the Report, and also means that the sequence alignments and phylogenetic trees, upon which taxonomic decisions often depend, are available as resources for all to use. A two-page summary of each Report chapter is published as an Open Access article by the Journal of General Virology as a Virus Taxonomy Profile. It remains a challenge to make these freely available resources comprehensive because of the pace of taxonomic change; over the last decade the number of virus families has almost doubled from 87 to 168. In meeting this challenge the ICTV will continue to rely on the enthusiasm, expertise and efforts of virologists from around the world.
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Polyesterase activities in bacterial isolates from seaweed and sponges, with potential utility in polyethylene terephthalate plastic and nanoparticle hydrolysis
More LessMany marine bacteria have evolved to produce a range of extracellular enzymes which facilitate their growth and survival in the harsh, oligotrophic conditions often present in marine environments. Marine sponge derived Streptomycesstrains have previously been reported to produce polyesterase enzymes, which are of interest for several biotechnological applications, including polyethylene terephthalate (PET) plastic hydrolysis.
Bacteria isolated from sponges and seaweed were screened for polyester hydrolysis activities using plate-clearing assays. Lipolytic and polyesterolytic activities were initially identified by employing tributyrin and polycaprolactone diol agar-based assay systems, respectively. Polyesterase activity was subsequently confirmed on both polycaprolactone and on PET-nanoparticle agar plates, resulting in the prioritisation of six isolates for Illumina next-generation genome sequencing.
These include three Bacillusspp., isolated from the brown seaweed Ascophyllum nodosum,and from marine lake sponges Stelligera stuposaand Eurypon major, together with a Maribacterstrain again isolated from S. stuposa, and Brachybacteriumsp. and Micrococcussp. isolates of deep-sea sponges Pheronemasp. and Inflatella pellicula, that were sampled at depths of 2129m and 2900m, respectively. Genome mining and comparative genomic analysis of these isolates is currently underway to identify genes encoding the observed activities and to assess homology with known PET hydrolases.
Microbes found living in association with filter-feeding sponges may have increased exposure to the plastics and microplastics that widely contaminate our marine ecosystems, thus representing a promising source of degradative activities towards synthetic polymers that could contribute to new plastic waste management strategies.
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Genomic and Phenotypic Characterisation of Multi-Drug ResistantEscherichia coli carried by meat handlers and slaughterhouse meat from North-Western Tanzania
Our research is part of a larger project Supporting the National Action Plan on Antimicrobial Resistance (SNAP-AMR) in Tanzania. The aspect of this work is to determine the AMR carriage and associated mobile genetic elements of extended spectrum β-lactamase (ESBL) producing Escherichia coli within the community of Mwanza, in North-Western Tanzania. From 54 slaughterhouses a total of 322 E. coli isolates were obtained from raw meat, with 210 originating from meat handlers. Initial antimicrobial susceptibility testing identified 71 (22%) and 27 (13%) of the meat and meat handler isolates, respectively, as being ESBL E. coli. ST2852 was the most abundant MLST, followed by globally disseminated and ESBL associated ST38. Whole genome sequencing and phenotypic analysis revealed 88% of our isolates were resistant to three or more antimicrobial classes. On average, our isolates carried at least 10 different AMR associated genes, with 80% having one or more AMR conferring chromosomal mutation. In relation to ESBL, 88 out of 98 of the isolates carried the globally disseminated allotype blaCTX-M-15, with over 55% of the isolates encoding blaCTX-M-15on multi-drug resistant (MDR) plasmids. The remaining strains encoded one of three different blaCTX-Mallotypes. Our data highlights high prevalence of MDR ESBL E. coli within the community and its potential risk of transmission through the food chain. In our future work, we will combine these community carriage data with our work on ESBL Enterobacteriaceae carriage and infection within healthcare settings, to evaluate the genomic epidemiology of ESBL organisms in this region.
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Development of a CRISPR interference system in Campylobacter jejuni
More LessCampylobacter spp. are the leading cause of bacterial food-borne illness in humans worldwide, with Campylobacter jejuniresponsible for 80% of these infections. There is no current vaccine and antibiotic resistance is emerging. There is an urgent need to understand fundamental C. jejuni biology for the development of new strategies to prevent and treat infections. The range of molecular tools available to regulate gene expression in C. jejuni is limited, which impacts studies into the function of essential and conditionally essential genes. My project aims to address this by applying a CRISPR-based interference system known as CRISPRi in C. jejuni as a means to control gene expression and thereby investigate gene function. To validate the CRISPRi system in C. jejuni, I have paired the dCas9 and sgRNA backbone from the Streptococcus pyogenesCRISPRi system with several C. jejuni-derived promoters to develop a series of CRISPRi constructs targeting several genes. Through rigorous sgRNA target design I have successfully targeted and repressed expression of the endogenous arylsulphatase (AstA) enzyme, as well as achieving partial repression of expression of the regulatory flagellar protein FlgR in two clinically relevant C. jejuni strains. This is the first report of a CRISPRi system for Campylobacter.
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Longevity and neutralisation activity of secretory IgA following SARS-CoV-2 infection
The mucosal barrier is a primary defence against inhaled pathogens, comprising secretory antibodies which have the potential to block viral entry and neutralise infection. There is an ongoing need for greater understanding of the mucosal immunity to SARS-CoV-2 infection. In this study, we investigated mucosal IgA through non-invasive saliva sampling of healthcare workers.
A total of 551 saliva samples were collected from staff at Great Ormond Street Children’s Hospital who previously tested positive for COVID-19. Participant metadata included age, gender, ethnicity and symptoms. IgA titres were measured by ELISA against viral antigens spike protein, nucleocapsid protein, and spike receptor-binding domain. SARS-CoV-2 neutralisation was measured using a VERO E6 cell culture infection assay.
We found that approximately 30% of saliva samples contained detectable IgA specific for at least one of the SARS-CoV-2 antigens. IgA levels in saliva decreased with the time post-infection, and were largely undetectable after six months. IgA titres specific to SARS-CoV-2 were lowest in participants over 60 years old. Specific saliva samples were identified which effectively neutralised SARS-CoV-2 virus infection of epithelial cells.
Our results suggest secretory IgA specific to SARS-CoV-2 can be detected in saliva following infection, an accessible sample type for testing, although titres decreased over time. Some saliva samples were able to neutralise SARS-CoV-2 infectivity against cultured epithelial cells. This data could be used to assess the risk of re-infection with SARS-CoV-2, as well as accelerate efforts to develop effective mucosal vaccination with longer lasting protection.
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Host mediated microbiome selection to study the changes in the nutrient cycling, root exudation and microbial population in the rhizosphere of soybean genotypes
More LessThe study was conducted with an aim to understand the changes in soil enzyme activity in the plant rhizosphere during host mediated selection of rhizosphere microbiome. Soil samples were collected from three different locations on a farm in the Reading area, UK. Two popular genotypes of Soybean (Glycine max) were chosen for the study, Kenchen and Siverka. Initial microbiome inoculum was prepared by mixing field soil with sterilized water and this was added to the autoclaved coir: sand mix. After four weeks of plant growth, plants were uprooted from the soil, rhizosphere soil collected for soil enzyme estimation and NGS analysis. Root exudates were collected and stored in freezer for analysis.
Growth of plants in legume soils was significantly higher. The plants in legume soil produced nodules which confirmed the presence of rhizobia in these soils. Independent analysis within each soil type showed that, the interaction between soil type and genotype is very significant with respect to enzyme activity. The presence of microbiome along with plants had a significant effect on enzymatic activity. GCMS analysis of root exudates from plants in each soil showed variations in individual chromatograms. The difference in enzyme activity with plant growth shows that there are changes taking place in the soil. Further in this study, NGS analysis of rhizosphere soil will shed more light into the changes happening in the soil microbiome and if the changes are related to the enzymatic changes and plant root exudation.
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Antibacterial Action of Visible 405-nm light for Bacterial Reduction in Blood Plasma
The introduction of risk prevention measures, such as blood screening and donor deferrals have dramatically reduced the incidence of transfusion-transmitted viral infections. Nevertheless, bacterial contamination of blood transfusion products remains a concern to patient health, and a range of pathogen reduction technologies have been developed to reduce this risk. Visible violet-blue light, in the region of 405-nm, has recently demonstrated potential for in situ treatment of ex vivo stored plasma and platelet products, without the need for additional photosensitizers.
This study assessed the broad-spectrum efficacy of 405-nm light against a range of bacteria implicated in transfusion-transmitted infections: Staphylococcus aureus, Staphylococcus epidermidis, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniaeandYersinia enterocolitica. Plasma was seeded with clinically-relevant low-level bacterial contamination (102-103CFUmL-1) and exposed to a 405-nm light dose of 360 Jcm-2 (1-hr at 100mWcm-2) using a small-scale exposure system. Broad spectrum antibacterial efficacy was observed, with 99.0 – 100% inactivation achieved for all bacterial species tested. Bacterial inactivation tests were then scaled-up to expose large volumes of prebagged plasma seeded with S. aureusat ~103 CFUmL-1, to 22mWcm-2 405-nm light, under agitation, (≤396 Jcm-2). Successful bacterial inactivation was observed using the large-scale exposure system, with a dose of 238 Jcm-2 (3-hr at ~22mWcm-2) achieving complete (3.5-log10) reductions in prebagged bacterial-seeded plasma (P=0.001). Results from this study support further development of visible 405-nm light technology as a bactericidal tool for application in transfusion medicine.
This abstract reflects the views of the author and should not be construed to represent FDA’s views or policies.
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Comparison of genome-derived and phenotypic antimicrobial resistance profiles of Shigella species isolated from patients with symptoms of gastrointestinal disease in England, 2015-2020
More LessIncreasing antimicrobial resistance (AMR) in Shigellaspecies is a global public health problem. We compared genotypic and phenotypic methods for the detection of AMR in Shigellaspecies to evaluate the use of genome data for surveillance and monitoring of emerging AMR.
Whole genome sequencing (WGS) data from 388 isolates of all four Shigella species were analysed for the presence/absence of specific AMR determinants and selected accordingly. Phenotypic antimicrobial susceptibility testing was performed using in-agar dilution on all viable and pure isolates (n=358). The genotypic and phenotypic AMR profiles were then compared.
There were 335 (93.6%) isolates resistant to at least one antimicrobial and 222 (62%) isolates were multi-drug resistant, of which the majority (77%) were associated with foreign travel. Out of a possible 2864 isolate/antimicrobial class combinations, we identified 119 unexpected results, giving an overall concordance of 96.8% between the two methods. There were 54 samples that had an AMR determinant expected to confer resistance that were phenotypically susceptible, of which 31/54 (57.4%) were associated with tetracycline resistance and trimethoprim-sulfamethoxazole resistance. There were 65 that were phenotypically resistant to a specific antimicrobial class, but no AMR determinant was detected, of which 32/65 (49.2%) were associated with isolates harbouring a single gyrA mutation and exhibiting an unexpectedly high minimum inhibitory concentration (MIC) to ciprofloxacin.
Although comparisons between both methods showed good correlation between the genotypic and phenotypic AMR profiles, phenotypic monitoring is required to identify novel AMR mechanisms and to update reference database used for WGS analysis.
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How do Streptomyces coordinate DNA repair and cell division following DNA damage?
More LessDNA damage often results in a pause of cell division until damage is repaired. In bacteria, a widely conserved response to DNA damage is the SOS response which relies on two proteins: the multifunctional recombinase RecA and the transcriptional repressor LexA. Under DNA-damaging conditions, this response activates proteins involved in DNA repair and the inhibition of cell division which in most unicellular bacteria results in temporarily filamentous growth. However, it is unknown how naturally filamentous growing bacteria like Streptomyces cope with DNA damage and how DNA damage repair is coordinated with cell division.
To identify novel regulators of cell division in Streptomyces that specifically function during DNA damaging growth conditions, we investigated the global response of Streptomyces venezuelae to several genotoxic agents, including mitomycin C, ciprofloxacin and methane methylsulfonate. To this end we have performed ChIP-seq experiments to identify the LexA regulon in Streptomycesandconducted RNA-seq experiments to determine the global response to DNA damaging agents in the wildtype and a recA mutant. The combined analysis of the available data sets has allowed us to obtain a comprehensive overview about the genes involved in the SOS response in Streptomycesand further analysis will enable us to understand how damage repair and cell division are coordinated in these bacteria.
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Meteorites: beneficial or toxic for life on Early Earth? Growth of an anaerobic microbial community on a carbonaceous chondrite
More LessMeteoritic material accumulated on the surface of the anoxic Early Earth during the Late Heavy Bombardment around 4.0 Gya. These meteorites may have provided the Earth with extra-terrestrial nutrients and energy sources for early life. How could the presence of meteorites have affected the origin and evolution of early life on Earth? And what is the influence of geothermal activity on the Earth’s surface? This research investigates the growth of an anaerobic microbial community from pond sediment on non-pyrolyzed (pristine) or pyrolyzed (heat-treated) carbonaceous chondrite ‘Cold Bokkeveld’. A microbial community was grown anaerobically in batch cultures containing a liquid environment and powdered non-pyrolyzed or pyrolyzed Cold Bokkeveld. Cell concentrations were measured by Colony-Forming Units on agar plates. The community composition in the presence of non-pyrolyzed meteorite was determined by 16S rRNA amplicon sequencing. Non-pyrolyzed Cold Bokkeveld supported the growth of a stable, anaerobic community containing mainly the Deltaproteobacteria Geobacteraceae and Desulfuromonadaceae. Members of these families are known to use elemental sulfur and ferric iron as electron acceptors, and organic compounds as electron donors. Pyrolyzed Cold Bokkeveld however, was inhibitory to the growth of the microbial community. These results show that carbonaceous chondrites can host an anaerobic microbial community, but that pyrolysis, e.g. by geothermal activity, can inhibit microbial growth and potentially toxify the material. This indicates that extraterrestrial meteoritic material and the environment on Early Earth could have shaped the nature of early microbial ecosystems by enhancing growth of microorganisms with metabolic capabilities favored in the presence of this material.
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Heterogenous susceptibility to R-pyocins in populations of Pseudomonas aeruginosa sourced from cystic fibrosis lungs
More LessBacteriocins are proteinaceous antimicrobials produced by bacteria which are active against other strains of the same species. R-type pyocins are phage tail-like bacteriocins produced by Pseudomonas aeruginosa. Due to their anti-pseudomonal activity, R-pyocins have potential as therapeutics in infection. P. aeruginosa is a Gram-negative opportunistic pathogen and is particularly problematic for individuals with cystic fibrosis (CF). P. aeruginosa from CF lung infections develop increasing resistance to antibiotics, making new treatment approaches essential. P. aeruginosa populations become phenotypically and genotypically diverse during infection, however, little is known of the efficacy of R-pyocins against heterogeneous populations. R-pyocins vary by subtype (R1-R5), distinguished by binding to different residues on the lipopolysaccharide (LPS). Each type varies in killing spectrum, and each strain produces only one R-type. To evaluate the prevalence of different R-types, we screened P. aeruginosastrains from the International Pseudomonas Consortium Database (IPCD) and from our biobank of CF strains. We found that (i) R1-types were the most prevalent R-type among strains from respiratory sources; (ii) there are a large number of strains lacking R-pyocin genes, and (iii) isolates collected from the same patient have the same R-type. We then assessed the impact of diversity on R-pyocin susceptibility and found a heterogenous response to R-pyocins within populations, likely due to differences in the LPS core. Our work reveals that heterogeneous populations of microbes exhibit variable susceptibility to R-pyocins and highlights that there is likely heterogeneity in response to other types of LPS-binding antimicrobials, including phage.
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Phage display strategy against outbreaks of unknown pathogens
More LessThe experience with SARS-CoV-2 that spread rapidly throughout the world makes us realize we need protocols to act quickly against unknown pathogens. The immune system protects recovering patients from any pathogen by producing antibodies against their immunogenic epitopes. Therefore, the strategy presented here is based on convalescent blood samples and a phage display platform for antibody and peptide drug discovery. Peptide libraries are screened against purified convalescent antibodies to identify immunogenic epitopes of the pathogen. Furthermore, the B cells of the recovery patients are used to amplify variable domains of antibody heavy and light chains expressed during the infection. These domains are cloned in a phagemid and produce free phage particles expressing the antibody fragments on their surfaces to select binders to pathogen immunogenic epitopes. These findings are essential in the identification of the unknown pathogen and the design of therapeutic molecules. In conclusion, this report describes a phage display strategy to combat outbreaks of unknown pathogens such as SARS-CoV-2.
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Genomic epidemiology of the first London outbreak of antimicrobial resistant sexually transmitted shigellosis
More LessShigellosis is an intestinal infection caused by Shigella bacteria. Shigella cause an estimated ~200,000 global deaths annually. Antimicrobial resistant (AMR) shigellosis is a significant cause of morbidity in high-income nations, with both multidrug resistant (MDR) and extensively drug resistant (XDR) cases being increasingly reported in Australia, England, and the USA. Sexually transmissible shigellosis was first described in San Francisco, 1974, but it would be a further 30 years before its first description in England. In 2004, London experienced an outbreak of Shigella sonnei (S. sonnei) mediated sexually transmitted shigellosis, associated with men-who-have-sex-with-men (MSM). Since then, sexually transmissible shigellosis has become endemic in England, with a greater than two-fold increase in Shigella diagnoses within sexual health services from 2015 to 2019. Through genomic exploration of samples from the original 2004 outbreak (provided by Public Health England (PHE)), we identified that the 2004 London outbreak isolates clustered within the base of genotype 3.1, lineage III, a lineage which has since gone on to dominate the global epidemiology of S. sonnei. The isolates displayed early evidence of varying degrees of antimicrobial resistance to several drug classes: macrolides, tetracyclines, beta-lactams and sulphonamides. Reconstructing the chronological process of how shigellosis has arrived at its current position in AMR and transmissibility is critical. Further investigation is underway to link this outbreak with MSM-associated shigellosis outbreaks occurring in the early 2000s in other countries to establish whether this lineage globally disseminated; determine the timeframe for global connectivity of shigellosis; and examine the outbreak isolates for virulence determinants.
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Biotransformation of phthalate plasticisers and bisphenol A by marine and freshwater fungi
More LessPhthalate esters (PEs) are environmentally ubiquitous micropollutants that are used as plasticizers and additives in diverse consumer products. Considerable concern relates to their reported xenoestrogenicity and the microbial-based attenuation of environmental PE concentrations is of interest to combat harmful downstream effects. Fungal PE catabolism has received less attention than that by bacteria, and particularly marine fungal species remain largely overlooked in this respect. We have compared the biocatalytic and biosorptive removal rates of di-n-butyl phthalate (DBP) and diethyl phthalate (DEP), chosen as two environmentally prominent PE representatives (exhibiting differing structures and hydrophobicities), by marine- and freshwater fungal strains. Bisphenol A, both an extensively used plastic additive and prominent environmental xenoestrogen, was included as a reference compound due to its previously well-documented fungal degradation. Partial pathways for DBP metabolization by these ecophysiologically diverse ascomycetes were proposed with the help of UPLC-QTOF-MS analysis. Species-specific biochemical reaction steps contributing to DBP metabolism were also observed. The involved reactions include initial cytochrome P450-dependent monohydroxylations of DBP with subsequent further oxidation of related metabolites, de-esterification via either hydrolytic cleavage or cytochrome P450-dependent oxidative O-dealkylation, transesterification, and demethylation steps - finally yielding phthalic acid as a central intermediate in all pathways. Beyond previous research into fungal PE metabolism which emphasises hydrolytic de-esterification as the primary catabolic step, a prominent role of cytochrome P450 monooxygenase-catalysed reactions is established.
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Non-typeable Haemophilus influenzae biofilm formation on primary human airway epithelia cultured at air-liquid interface
IntroductionPrimary ciliary dyskinesia (PCD) is an inherited heterogeneous disorder associated with defective motile cilia on airway epithelia, resulting in impaired mucociliary function and ultimately, bronchiectasis. Non-typeable Haemophilus influenzae (NTHi) is frequently isolated from PCD airways where it resides in biofilms. It is unclear if biological pathways in response to NTHi infection differ in PCD, contributing to increased susceptibility to biofilm-associated infection.
AimThe development of a biologically representative co-culture model to investigate the interactions between PCD airway epithelia and NTHi biofilms.
MethodsAir-liquid interface (ALI)-cultured non-PCD primary nasal cells were grown in transwells. Barrier properties were assessed by trans-epithelial electrical resistance (TEER), FITC-dextran passage and tight junction staining. Ciliary function was assessed by high-speed video microscopy at 37°C. ALI cultures were infected with a PCD NTHi isolate at multiplicity of infections (MOIs) 10, 50 or 100 and co-cultured to form biofilms over 3 days. TEER was measured pre- and post-infection, NTHi recoverability assessed by conventional culture, and biofilm formation confirmed by scanning electron microscopy (SEM).
ResultsEpithelial culture at ALI was successful, with tight barriers being formed and normal ciliary beat pattern and frequency recorded (mean 14Hz). Following co-culture, TEER increased relative to pre-infection at all MOIs but maximum NTHi recoverability was at MOI 50 (median colony forming units, 6.8x106per cm2). SEM confirmed NTHi biofilm formation at MOI 50.
ConclusionsA successful NTHi biofilm co-culture model on human primary epithelia was established. This will be used with PCD epithelia to investigate host-pathogen interactions during NTHi biofilm colonisation.
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Systematic Reconstruction of the Complete Two-Component Sensorial Network in Staphylococcus aureus
In bacteria, adaptation to changes in the environment is mainly controlled through two-component signal transduction systems (TCSs). Most bacteria contain dozens of TCSs, each of them responsible for sensing a different range of signals and controlling the expression of a repertoire of target genes (regulon). Frequently, TCS control key physiological changes required for pathogenesis and/or antimicrobial resistance. Over the years, identification of the regulon controlled by each individual TCS in different bacteria has been a recurrent question. However, limitations associated with the classical approaches used have left our knowledge far from complete. In this report, using a pioneering approach in which a strain devoid of the complete nonessential TCS network was systematically complemented with the constitutively active form of each response regulator, we have reconstituted the regulon of each TCS of S. aureus in the absence of interference between members of the family. Transcriptome sequencing (RNA-Seq) and proteomics allowed us to determine the size, complexity, and insulation of each regulon and to identify the genes regulated exclusively by one or many TCSs. This gain-of-function strategy provides the first description of the complete TCS regulon in a living cell, which we expect will be useful to understand the pathobiology of this important pathogen.
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Role of enteroaggregative E.coli induced activation of epidermal growth factor receptor in cultured human intestinal epithelial cell lines
More LessEnteric pathogens exploit the versatility of cytoskeletal system for internalization into non-phagocytic cells, as a crucial step in their pathogenic life cycle. Enteropathogenic Escherichia coli(EPEC) andEnterohemorrhagic Escherichia coli (EHEC) were shown to form actin pedestals in host cells using type-III secretion system. Earlier, we reported that EAEC induced increase in intracellular calcium ions might have crucial role in F-actin rearrangements in INT-407 cells leading to its invasion. It was suggested that EGFR might contribute in Rck-mediated adherence and invasion of Salmonella in host cells. In the present study, we assessed the role of EAEC induced activated EGFR in human intestinal epithelial cell lines (INT-407 & HCT-15). The presence of activated EGFR was detected in membrane fractions ofeach cell line, infected with two different strains of EAEC (EAEC-T8 & EAEC-O42) separatelyfor 3h in presence and absence of Tyrphostin AG1478 (EGFR-inhibitor), by western immunoblotting using p-EGFR (Y1068) antibody. Adherence and invasion of EAEC-T8 to each cell line were checked in presence of Tyrphostin AG1478. Further, the effect of Tyrphostin AG1478 on cytoskeletal F-actin rearrangement of EAEC-T8 infected cells was assessed under confocal microscope following staining with TRITC-phalloidin. EAEC-T8 induced maximum increase in EGFR autophosphorylation at Y1068. Adherence and invasion of EAEC-T8 as well as this organism induced cytoskeletal F-actin polymerization were found to be inhibited in presence of Tyrphostin AG1478. Our study revealed that EAEC induced activated EGFR might play a major role in host cell adherence and cytoskeletal rearrangements leading to invasion of the organism in these cells.
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