- Volume 4, Issue 5, 2022

Metals 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.

The 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.

Ralstonia 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.

Bioprospecting 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.

Polymicrobial 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.

Despite 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.

Antibiotic 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.

The 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.

Tuberculosis 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.

Staphylococcus 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.

In 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.

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.

Increasing 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.

Trimethylamine 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.

Microbial 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.