- Volume 1, Issue 1A, 2019

Pediocin PA −1 is class IIa bacteriocin that displays efficient antimicrobial activity against pathogenic Listeriaspp. This bacteriocin is known to lose activity during long periods of storage especially at non optimal pH, thus reducing its usefulness for the pharmaceutical and food industries. Loss of activity has been attributed to oxidation of the methionine residue at position 31, however, replacement of this residue by leucine results in a peptide with activity equivalent to that of the native peptide. In this work, the heterologous expression of the structural (with Met31 to Leu substitution), accessory and transport genes from pediocin PA-1 operon was carried out in Escherichia coli TunerTM (DE3) cells. The sequences of all genes were redesigned using codon bias for the host and were cloned into an expression vector that allows control of plasmid copy number. The heterologous expression of pediocin Met31Leu was optimized for temperature, induction time, IPTG concentration and plasmid copy number and was evaluated via antimicrobial activity assays against Listeria innocua DPC3572. Maximum activity (2560 AU mL−1) was achieved using low plasmid copy number and 6 h of induction at 37 °C with 1 mM of IPTG. Recombinant pediocin PA-1M31L was successfully purified in 5 steps (>95 % purity) as confirmed by mass spectrometry (4606.27 Da) with a yield of 0.725 mg per liter of culture. This variant showed a similar spectrum of activity to the native pediocin PA-1 and is an interesting alternative for industrial applications due to its greater stability.

Dimer formation is a serious threat to the stable maintenance of ColE1-like plasmids. Dimers form infrequently by homologous recombination but accumulate rapidly by having two origins of replication. This results in elevated plasmid loss and a reduction in host cell growth rate. Plasmid dimers are resolved to monomers by the XerCD recombinase plus accessory proteins ArgR and PepA, acting at the cer recombination site. The circular chromosome of E. coli also forms dimers infrequently, and consequent failure of chromosome partition results in filamentation, SOS induction, and failure of cell division. Site-specific recombination is required for dimer resolution during cell division in a process facilitated by XerCD acting at the dif (deletion induced filamentation) site near the E. coli chromosome terminus. ArgR and PepA accessory proteins are nonessential, but the septum-associated protein FtsK is necessary for dimer segregation, suggesting the XerCD/difcomplex interacts with division septums. Our preliminary work had revealed homology between cer and a 170 bp chromosomal site (tcs, terminal region cer-like site) 1.2 min from dif. The tcs site and surrounding region was cloned into plasmids, dimer plasmids were purified and then assayed for tcs-mediated recombination. Our results demonstrate that a construct with a 500 bp tcs insert supports XerCD-mediated recombination, whereas smaller constructs were recombination-deficient. The absence of plasmid monomerization in mutant strains indicates that ArgR and PepA are required for recombination. Additionally, the tcs knockout strain displayed moderate filamentation of cells. Given the similarities between cer, tcs, and dif, these results suggest that tcs could facilitate chromosome dimer resolution.

While transcriptional reprogramming is perhaps the most well understood form of controlling gene expression in response to nitrogen starvation in bacteria, how post-transcriptional regulation (PTR) of gene expression contributes to this adaptive response remains elusive. Small regulatory RNAs (sRNAs) are the major post-transcriptional regulators of gene expression in bacteria. They regulate gene expression by base pairing to target mRNAs, leading to enhanced translation or inhibition of translation and/or alteration of mRNA stability. To form productive interactions with target mRNAs, most sRNAs require an RNA chaperone. In many bacteria of diverse lineages, the RNA chaperone Hfq plays a central and integral role in the PTR of gene expression by stabilising sRNAs and promoting their interactions with cognate mRNAs. Comparative analysis of the transcriptomes of Escherichia coli at different stages of nitrogen starvation reveal that levels of sRNA vary throughout starvation. We used Hfq as a surrogate to study sRNA-mediated PTR of gene expression during sustained nitrogen starvation. Our results indicate that sRNAs-mediated PTR of gene expression plays a major role in the adaptive response to sustained nitrogen starvation. Intriguingly, using single-molecule PALM, we reveal that Hfq is involved in the formation of intracellular structures which functionally might resemble processing (P) bodies found in eukaryotic cells involved in mRNA turnover.

Human nasal colonization with Staphylococcus aureus sets the stage for subsequent systemic infection. The mechanisms responsible for colonization are not fully understood. This study characterizes S. aureus adhesion to nasal mucosa in vitro and observes the interaction of S. aureus with mucin. S. aureus showed significantly higher binding to mucin during stationary phase in comparison to log-phase cells. Adherence of S. aureus srtA mutant to mucin was not significantly different from wild-type, thus Srt A shows no influence on S. aureus interaction with mucin. Adherence to mucin was saturable in a dose- and time-dependent fashion. Biotin–labelled mucin bound to surface protein (55 kDa) of cell wall extracted S. aureus which is encoded by the sbi gene. These data suggest that adherence factors are present on the surface of S. aureus such as sbi. Purified recombinant Sbi was prepared and the mucin binding capacity of the protein was tested by ELISA. In order to determine the function of specific domains of Sbi in adhesion, Sbi constructs with, without the IgG-domain and with B2-glycoprotein domain were expressed on the surface of E. coli BL21(DE3). The expression of Sbi with the B2-glycoprotein domain on the surface adhesion, emphasizing the role of Sbi in mucin adhesion. A profound binding effect was observed with Sbi incubated in wells coated with host mucin. Therefore, it is proposed to investigate a novel interaction of S. aureus to host mucin in order to control S. aureus nasal colonization.

Actinobacteria have a protein O-glycosylation system that resembles eukaryotic protein O-mannosylation. Both M. tuberculosis and S. coelicolor have growth retarded phenotypes when protein-O-mannosyl transferase (Pmt), which transfers mannose from polyprenol phosphate mannose to a target protein, is absent. Moreover, S. coelicolor pmt- mutants are resistant to φC31 phage infection and have increased susceptibility to vancomycin and multiple b-lactams. S. coelicolor strains that lack polyprenol phosphate mannose synthase (Ppm1), which transfers mannose from GDP-mannose to polyprenol phosphate, are even more susceptible to antibiotics and a ppm1- mutant in M. tuberculosis is lethal. Pmt and Ppm1 are therefore possible new targets for the isolation of antimicrobials to be used against M. tuberculosis. Our aim is to further understand the structure and function of these enzymes. Sequence alignments and structural bioinformatics were used for S. coelicolor Ppm1 and Pmt to identify site-directed mutagenesis targets. Mutant alleles were introduced into ppm1- (DT3017) or pmt- (DT2008) S. coelicolor strains using conjugative integrative plasmids and scored for their ability to complement phage sensitivity and antibiotic hyper-susceptible phenotypes. Twenty-two highly conserved Pmt residues were each changed to alanine and six mutants failed to complement DT2008, indicating essentiality. Modelling these six residues indicated that five are positioned close to the predicted catalytic DE motif. For Ppm1, ten mutant alleles were tested and eight were essential for DT3017 complementation, with four residues positioned close to the predicted catalytic DXD motif. Whilst some of the mutations were predicted to impair catalytic activity, others may have affected localisation or substrate binding.

We previously identified the diversity of antimicrobial activity in nasal microbiomes that correlates with presence and absence of Staphylococcus aureus. The major competitor of skin surfaces is Staphylococcus epidermidis that can express diverse antimicrobial activities. Ten-fold less frequently found in skin and nasal microbiomes, Staphylococcus hominis is relatively unstudied. The aims of the research were to develop insights into the factors of S. hominis that contribute to the dynamics of competition with these major skin colonising staphylococci. One prominent inhibitory strain, S. hominis was selected to culture with both S. aureus SH1000 and S. epidermidis with aim to use genome sequencing to reveal loci contributing to competition. S. aureus evolved during the competition of S. epidermidis and inhibitor-producing S. hominis to reveal two discrete phenotypes. Non-pigmented clone expressed less alpha-haemolysin and had a SNP in agr Cencoding the receptor of the Agr quorum-sensing system. More Pigmented clone had a SNP in sig Bencoding the accessory sigma factor required for expression of the staphyloxanthin. Distinctively, competition of S. aureus SH1000 with S. epidermidis revealed evolution of S. aureus that corresponded with a SNP in the lytSgene of the LytSR two-component system controlling murein hydrolase activity and autolysis. Pacbio genome sequencing followed by use of AntiSMASH revealed the S. epidermidis inhibitory strain, which enabled persistence of S. hominis, encoded the lantibiotic gallidermin biosynthesis operon on a 39 kb plasmid. Gallidermin decreased S. aureus survival to competitor S. epidermidis, and an increased ability of S. hominis to maintain its population size during evolution experiments, which supported the dynamic relationship of the three staphylococci. Future studies will unravel explanations for the contributions the identified loci make to multi-species competition.

Bacterial biofilms are highly complex communities, composed of highly structured extracellular polymers and subpopulations of differentiated cells, such as persisters. These contribute to the resistance of bacterial biofilms to antibiotics, creating a significant issue in the treatment of infections and resulting in elevated levels of mortality and morbidity. Here, we use a microfluidic system coupled with time-lapse microscopy and fluorescent dyes for exopolysaccharide (EPS) and extracellular DNA (eDNA) to investigate how the architecture of the growing biofilm is ordered. We show that in Pseudomonas aeruginosa EPS is deposited first in the initial stages of microcolony development, but that eDNA then acts as a leading edge for further microcolony expansion. We explore how this assembly is perturbed by the introduction of different antimicrobial agents. Further, working with partners in the European Association of National Metrology Institutes (EURAMET) we are developing cross platform methods for the label-free localisation of antimicrobial agents and bacterial components within the biofilm. These platforms include 3D OrbiSIMS (secondary ion mass spectrometry) and Raman spectroscopy. Here we demonstrate the localisation of key biofilm components such as Pseudomonas Quinolone Signal (PQS) molecules in a 3D chemical map of the biofilm.

Gut eukaryome refers to the collection of fungi and protists in the gut. Until recently, all eukaryotic gut microbes were considered parasites and subject to elimination. Though this is true in some instances, critical evaluation of the literature reveals that most microbial eukaryotes are harmless often colonizing the human gut for long periods of time. Evidence is accumulating that the eukaryome plays important ecological roles in gut communities, as well as, in health and disease of the host. Nonetheless, systematic examination aiming to obtain baseline information of their prevalence and diversity in human populations is lagging. To address this knowledge gap, we collected fecal samples from a population of adults residing in north Thailand (n=211), who showed no gastrointestinal (GI) symptoms and had no history of GI diseases. We then examined the prevalence and diversity of two commonly found eukaryotic genera: the stramenopile Blastocystis (Blastocystis spp.) and the yeast Candida (Candida tropicalis and Candida albicans). Twenty three percent of individuals were positive for Blastocystis. Their sequences grouped in six of the nine clades that colonize humans. Twelve percent of the study population was positive for Candida, 4 % for C. albicans and 8 % for C. tropicalis, while concurrent colonization was also noted in some individuals. Eukaryotic bacterial interactions, as well as, interplay with diet and body mass index are also discussed. This is the first study providing data on the eukaryome of Thai populations and evidence that microeukaryotes traditionally considered as pathogenic asymptomatically colonize the gut of healthy humans.

Worldwide, hundreds of thousands of healthcare acquired infections (HAIs) are reported each year. Contamination of hospitals is a source of, and allows dissemination of HAIs. In healthcare settings one of the major vectors of contamination is healthcare workers’ uniforms. As surfaces become contaminated, bacteria can then be contacted by patients or staff who may indirectly spread bacteria to patients. Direct and indirect spread of bacteria could result in infection of patients and increased infection rates. Further consequences include increased levels of antibiotic use and costs. A pilot study was conducted at Antrim Area Hospital, Northern Health and Social Care Trust. 100 pre-shift and 100 post-shift healthcare workers’ uniforms were assessed for Staphylococcus aureus and Enterococcus spp. isolates. We found increased levels of antibiotic resistant S. aureus and Enterococcus spp. contamination on post-shift uniforms compared to zero to minimal contamination of pre-shift uniforms. A biobank of isolates was subsequently characterised for antibiotic sensitivity using European Union Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines – 51 % of S. aureus isolates were classed multi-drug resistant. Genomic diversity was assessed using Random Amplification of Polymorphic DNA (RAPD) – high levels of similarity was found amongst isolates. As one means of reducing uniform bioburden, we conducted analysis of a novel surface-active organisilane disinfectant named Goldshield (GS). GS was marketed as a long lasting antimicrobial prevent (re)contamination. GS technology displayed bactericidal, sporicidal and anti-biofilm properties in laboratory testing providing rationale for an intervention where GS could be incorporated into hospital laundry and assessed for potential use in infection control.

Diarrhoeal disease is a major public health concern, with over 500 000 childhood deaths recorded every year, (www.who.int). The leading cause of infantile diarrhoea is pathogenic Escherichia coli (EPEC), characterised by watery diarrhoea which varies in levels of severity. In recent studies a growing incidence of asymptomatic EPEC carriage has been recognised in Asia and the Middle East, suggesting the possibility that communities may act as a reservoir for this microorganism, (Kader, 2009; Alikhani, Mirsalehian and Aslani, 2018). Using a combination of molecular biology and bioinformatic analysis, we aim to characterise asymptomatic strains from these regions, determine their key virulence factors and find new ways of combatting this pathotype. At present we have developed a diagnostic multiplex PCR which incorporates known virulence genes of diarrhoeagenic E. coli pathotypes for quick and simple detection of EPEC. Additionally, we have optimised an infection assay using HeLa cells to phenotypically characterise typical and atypical colonisation and also identified bacteriocins capable of killing EPEC.

The presence of fungal species on the surface skin and hair is a known finding in many mammalian species and humans are no exception. Superficial fungal infections are sometimes a chronic and recurring condition that affects approximately 10–20 % of the world’s population. However, most species that are isolated from human tend to occur as co-existing flora. This study was conducted to determine the diversity of fungal species isolated from the hair and nails of workers in the central region of Saudi Arabia where there are not many observational studies on the mycological species. Male workers from Riyadh, Saudi Arabia were recruited for this study and samples were obtained from their nails and hair for mycological analysis which was done using Saboraud’s agar and sterile wet soil. Fungal isolates were examined microscopically. Twenty four hair samples yielded a total of 26 species from 19 fungal genera. Chaetomium globosum was the most commonly isolated fungal species followed by Emericella nidulans, Cochliobolus neergaardii and Penicillium oxalicum. Three fungal species were isolated from nail samples, namely, Alternaria alternata, Aureobasidium pullulans and Penicillium chrysogenum. Most of the isolated fungal species (17 of the 26 or 65.38 % of the isolated fungal species). Most of our isolated fungal species have not been thoroughly characterised nor morphologically classified.

Perkinsus species are important marine parasites of molluscs including mussels and oysters with substantial commercial and environmental impact. Perkinsus forms a sister lineage to the apicomplexan parasites (e.g. malaria agent, Plasmodium) and share similar invasion-related structures with these better studied pathogens. However, much less is known of the transmission and invasion biology of Perkinsus spp. We are developing genetic tools to study these parasites, and Perkinsus is emerging as an experimental model for marine parasites. The life cycle of Perkinsus species start with motile zoospores that are ingested by the host via filtration of sea water. After infection of the mollusc, these cells develop into a replicating non-motile vegetative form of the parasite, the trophozoite. Upon host death or morbidity, the parasites are released back into the water column where they differentiate into zoosporangia that release up to 100 motile zoospores. In vitro, Perkinsus species can be maintained as trophozoites in an axenic sea water-based media, and this is the best studied form of the parasite. However, Perkinsus olseni, can be triggered to sporulate in vitro using Ray’s fluid thioglycollate media, and we seek to better study this phase of the lifecycle that is essential for disease spread. To do this, we are determining stage-specific gene expression, during the transformation from trophozoites to zoospores, by RNA-Seq. This will both identify genes that are specific to this growth stage, and also identify zoospore-specific promoters that can be used in experimental manipulation and study of this poorly known cell stage.

Mastitis is a common disease in dairy cattle. An important aetiological agent of this disease is bacteria of the genus Streptococcus; hence, exploring the mechanisms of virulence in these bacteria is an extremely important step for the development of effective prevention programmes. The purpose of our study was to determine the ability to produce biofilm and the occurrence of selected invasiveness factors among Streptococcus isolated from cattle with the clinical form of mastitis in northeastern Poland. Most of the isolates analyzed demonstrated an ability to produce biofilm (over 70 %). Virulence genes were searched for in S. agalactiae, S. uberis and S. dysgalactiae. For S. agalactiae, only four genes were confirmed: rib (33 %), cylE (78 %), bca (37 %), and cfb (100 %). The genes pavA, scpB, bac and lmb were not present in any of the tested strains. The dominant serotypes were Ia (n=8) and II (n=8), in addition to some strains that were not classified in any of the groups (n=6). Out of the eight selected genes for S. uberis only one was not found (lbp). Finally, two genes were chosen for S. dysgalactiae (eno and napr), and their presence was confirmed in 76 % and 86 % of the strains, respectively. The experiment showed that strains of Streptococcus spp. isolated from dairy cattle with clinical cases of mastitis in the northeastern part of Poland possess several invasiveness factors that can substantially affect the course of the disease, and this should be considered when developing targeted prevention programmes.

Colibacillosis caused by Avian Pathogenic Escherichia coli (APEC) is a significant cause of morbidity, mortality and carcass condemnation to the poultry industry worldwide resulting in significant economic losses. We assessed a multiplex PCR for classifying diagnostic APEC and characterized these isolates using gene profile analysis.48 E. coli isolates collected between August and October 2018 through the Poultry Disease Research Center (PDRC) Diagnostic Laboratory were analyzed. Isolates were screened using multiplex PCR targeting genes associated with APEC chromosomal and plasmid virulence. Isolates were assessed for relationship between gene profile and host tissue of origin. Overall, isolates met the criteria for definition as well-developed pathogens with more than 90 % of isolates positive for the genes iroN, ompTp and hlyF; 78 % were positive for aerJ and 67 % for iss. A significantly lower prevalence was observed for cvaC, etsB, ireA and papC (range 5–36 %). When overall gene prevalence was examined for tissue of isolation, we found that APEC from the ovary, bone marrow, pericardium and lung had higher average numbers of genes compared to isolates recovered from skin and yolk sac. Genes associated with the ColV virulence plasmid (iss, iroN, hlyF and ompTp) were detected in 43 of 48 isolates (89.5%) further confirming the ColV plasmid is the defining trait of the APEC subpathotype. The use of a multiplex panel to screen for APEC has shown good correlation with pathogenesis, and tissue source and correlates well with invasive strains. Path panel diagnostics is available through PDRC, providing significant value to APEC screening.

Uneven flock growth (UFG) occurs when a broiler flock exhibits a wide range of bodyweights at slaughter. Automated chicken processing plants operate on an average bodyweight for the flock being processed so UFG can cause disruption to the processing flow that requires manual remediation. It is known that enteric virus infections of young broiler flocks lead to varying degrees of growth restriction that range from runts at hatch and severe early stunting, which are generally culled, to poor flock performance and UFG. In order to investigate the make-up of communities of enteric viruses (virome) associated with poor growth and also their infection timings and persistence in broiler flocks, a longitudinal survey of 7 commercial broiler flocks of varying performance over 3 successive broiler crops was undertaken with gut and faecal samples collected from 50 birds at each of 12 timepoints from pre-hatch to slaughter. The samples were pooled for each timepoint, then processed to enrich for viruses by removing host cells and bacteria. The RNA was extracted from each timepoint sample, amplified by whole transcriptome amplification followed by whole genome library preparation for the Illumina MiSeq platform and libraries multiplexed. Bioinformatics analysis used ViromeScan which facilitates metagenomic studies of quality-trimmed sequencing reads. The presence of diverse viral families was seen including the known major enteric viruses of the astrovirus, picornavirus, reovirus and parvovirus families. A greater diversity of viruses was observed in the flocks of poorer performance than in those flocks of good performance.

Dothistroma needle blight caused by D. septosporum has emerged in the British Isles as a major threat to Corsican pine, lodgepole pine and Scots pine. There is increasing evidence that mycoviruses can reduce the growth and pathogenicity of fungal plant pathogens. The aim of the present study is to characterise a double-stranded RNA virus found in D. septosporum and investigatefor putative hypovirulence, a common feature noted for mycoviruses, which might be used for biological control to invasion by more aggressive strains of the fungus. To this endthe viral genome was cloned and sequenced revealing four genomic segments, each one containing a single open reading frame (ORF) flanked by 5’ and 3’ untranslated regions. The ORFs encode the RNA-dependent RNA polymerase, the capsid protein, a protein of unknown function and a putative protease, respectively. Phylogenetic analysis of the sequences obtained revealed their similarity to members of the established family Chrysoviridae, genus Alphachrysovirus, which are encapsidated in isometric particles and are known to elicit hypovirulence in their hosts. Subsequently, virus-free and virus-infected isogenic lines were generated to determine any effects of the mycovirus on fungal fitness and pathogenicity. More specifically, the virus-infected isolate is currently being assessed in comparison to the virus-free one in terms of radial growth in solid culture, biomass in liquid culture, pathogenicity in pine trees and production of the mycotoxin dothistromin. In conclusion, this study reports the first mycovirus ever found in D. septosporum.

Clostridioides difficile, formerly known as Clostridium difficile, is a pathogen of increasing agricultural and clinical significance. This Gram-positive, anaerobic, toxigenic bacterium produces extremely robust spores which are highly resistant to environmental pressures, allowing this organism to persist in the environment for extended periods. C. difficile is capable of infecting both animals and humans. As a commensal organism it does not typically pose a threat to its host unless antibiotic treatment disrupts the normal gut flora. However following a decline in microbial diversity, C. difficile opportunistically colonises the host gut and proliferates, thereby causing C. difficile infection (CDI). CDI is transmitted via the faecal-oral route with spores surviving transit through the gastrointestinal tract to the gut. Symptoms of CDI range from mild diarrhoea to pseudomembranous colitis (PMC), toxic megacolon and death. C. difficile ribotypes present in farm animals have been found in workers tending to them, suggesting a potential zoonotic transmission. Sporulation is intrinsic to C. difficile’s transmissibility and the spores are highly resilient. Previous joint work at both the Queen’s University Belfast and Royal Victoria Hospital (Belfast) identified a non-sporulating variant of ribotype 078, the most prevalent ribotype infecting humans in Northern Ireland. Investigations into the prevalence of the non-sporulating variant in a range of animal and clinical derived ribotype 078 isolates are currently ongoing. Additionally, the presence of the non-sporulating variant in other toxigenic clades of C. difficile is undergoing assessment.

Brucella organisms are Gram-negative, intracellular facultative extracellular bacteria that infect a variety of animals. On March 2018, a pregnant dwarf sperm whale (Kogia sima), stranded and aborted on Herradura beach at the Pacific coast of Costa Rica. K. sima is a criptic species; very little is known of its biology and worldwide distribution, however it is still hunted in Asia. Brucella sp. was recovered from multiple tissues of the female and the calf, and examined by biochemical tests, MLVA-16, brucellader and HRM real time. The isolates were used for whole genome sequencing and the reads were aligned to Brucella abortus 9–941 as the reference, alltogether with other Brucella species. A total of 27 365 variable sites were extracted and a phylogenetic reconstruction by maximum likelihood was produced. The phylogenetic tree revealed that the K. sima isolates are related to Brucella sp. F5/99, a singular strain recovered on 1992 from a bottlenose dolphin captive in California, classified as sequence type (ST) 27 by multi-locus sequence type. This ST27 was described in Brucella isolates with zoonotic capacity and therefore transmission to humans from Peru and New Zealand. This is the first report of Brucella ST27 recovered from a host of the Eastern Tropical Pacific and of Brucella infection in a dwarf sperm whale. Our results shows that the range of marine mammals infected by Brucella sp. is wider than our current knowledge, and that biosafety measures should be increased when handling the stranded mammals, as the zoonotic transmission is of major risk.