- Volume 4, Issue 5, 2022

Per-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.

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.

Since 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.

Gene 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

Most 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.

Over 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.

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).

Bacteriocins 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.

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.

A 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.

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.

Many 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.

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.

Campylobacter 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.

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.

The 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.

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.

Increasing 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.