- Volume 1, Issue 1A, 2019

Studies have shown evidence of human exposure to aflatoxin M1 due to the consumption of contaminated milk and dairy products (mainly cheeses). This poses a great risk to public health, since milk and milk products are frequently consumed by a portion of the population considered immunosuppressed, children and the elderly. Knowledge of the negative impacts of aflatoxins on health and economics has led to investigations of strategies to prevent their formation in food, as well as to eliminate, inactivate or reduce the bioavailability of these toxins in contaminated products This study evaluated the effect of microbiological methods using lactic acid bacteria on aflatoxin M1 (AFM1) reduction in Minas Frescal cheese (typical Brazilian product, being among the most consumed cheeses in Brazil) spiked with 1 µg l−1 AFM1. Inactivated lactic acid bacteria (0, 5%, v/v de L. rhamnosus e L. lactis) were added during the cheese production process. Nine cheeses were produced, divided into three treatments: negative controls (without AFM1 or lactic acid bacteria), positive controls (AFM1 only), and lactic acid bacteria+AFM1. Samples of cheese were collected on days 2, 10, 20 and 30 after the date of production and submitted to composition analyses and determination of AFM1 by high performance liquid chromatography. The reductions of AFM1 in cheese by lactic acid bacteria at the end of the trial indicate a potential application of inactivated lactic acid bacteria in reducing the bioavailability of AFM1 in Minas frescal cheese without physical-chemical and microbiological modifications during the 30 day experimental period.

Listeriosis is an important food-borne disease responsible for high rates of morbidity and mortality. L. monocytogenes has been the cause of several food-borne outbreaks and product recalls throughout the world. It can adapt and survive in a wide range of stress conditions which makes it difficult for food producers to eradicate. The goal of this study was to use phenotypic assays and whole genome sequencing to elucidate possible links between food related stress resistance and virulence phenotypes in L. monocytogenes strains originating from different sources. Four L. monocytogenes isolates from sweetcorn and one isolate from a food processing environment (control) were sequenced and evaluated for the ability to survive in acid (pH 3.5, 15 min), in the presence of a commercial antimicrobial mixture (2 % v/v, 90 min), heat (60 °C, 5 min) and hydrogen peroxide (420 mM, 15 min). Results showed that the strains had different resistance levels to the above stressors with the environmental strain being more susceptible to heat and the commercial antimicrobial. Also, results showed that the four sweetcorn isolates were more virulent than the environmental isolate as they had significantly higher attachment and invasion capacity onto HCT-8 cells (P Pan-genome analysis revealed that the four isolates fall within a class associated with recent outbreak strains. Single Nucleotide Polymorphisms (SNPs) analysis was performed on the five genome sequences and subsequent cluster analyses on the resulting whole genome SNP matrix revealed differences between the strains.

Community profiling is one of the most utilised tools in microbial ecology today. The relationships between microbial communities and their environment affect ecosystem function in fields spanning from medicine to agriculture; and understanding the community dynamics of a microbial community is key to understanding the complex effects of human intervention. In this study, we look at the effects of long-term tillage and fertilization regimes in soils from an agricultural block-designed field trial set up in 2001. By studying the microbial community composition, absolute microbial abundance and diversity of denitrification functional genes in the context of environmental data, we were able to address the question of how specific land management histories affect the diversity and distribution of bacteria and denitrification genes within agricultural soils. It was found that microbial communities appear to be largely unaffected by land management history, and cluster predominantly by spatial location within the field, despite lack of significant environmental variation. In this well-established agricultural field trial, Euclidean distance is the major identifiable determinant of microbial community dissimilarity (as well as dissimilarity in microbial abundance). That ecological drift, rather than physicochemical factors can be the major determinant of genetic potential may have consequences for attempts to understand nutrient availability in agricultural systems. Additionally, the overwhelming variation caused by spatial distance indicates that block designed experiments may not always have sufficient statistical power to identify any effects of human treatment.

Vibrio vulnificus is a significant human pathogen found in high numbers in oysters. Despite the environmental prevalence of V. vulnificus, clinical cases are uncommon. We hypothesised that in vivo competition between V. vulnificus and other strains/species resulted in the killing of hypervirulent strains, reducing clinical incidence. To assess this, we have developed an oyster model into which we can ensure ingestion of high quantities of V. vulnificus using a defined ‘marine snow’. Marine snow describes the aggregation of naturally occurring phytoplankton, bacteria, debris and other organic materials. Our marine snow substrate is comprised solely of the diatom, Thalassiosira pseudonana. Bottles containing artificial seawater, 109 T. pseudonana, V. vulnificus culture and hyaluronic acid were rotated at 16 r.p.m. for 24 h to generate aggregates. V. vulnificus-containing marine snow was added to beakers holding individual oysters. Following 24 h uptake, oyster stomachs were excised and homogenised in PBS. The resulting suspension was serially diluted for plate-counts and DNA extracted for downstream qPCR analysis. Diatom-based aggregates present a controllable and reproducible model for incorporating V. vulnificus into marine snow. Using this methodology, we demonstrate greater uptake of V. vulnificus by oysters than any current study. This has potential applications for future in vivo work studying a range of microorganisms in oysters, such as other human pathogens or those of interest to aquaculture. Future work will undertake in vivobacterial competition assays to determine the role that intra-/inter-species competition has on the ecology of V. vulnificus and whether this impacts the clinical incidence.

Bacterial biofilm formation is an important survival strategy in multiple environments. It is affected by the attachment surface, the bacterial strain and the surrounding environment. In Salmonella enteritidis, a biofilm-forming foodborne pathogen, the molecular biofilm regulators act as on-off controls between the sessile and the planktonic population, while the exact underlying formation mechanism still remains unclear. The aim of this project is to study the effect of alkaline environment on the formation of Salmonella biofilms and to examine the architecture of biofilm produced under alkaline conditions, by use confocal microscopy. Neutral pH was found to be the optimal pH for Salmonella biofilm formation, while pH 10 significantly reduces it (P-value=0.015). However, cell viability remains high at pH 10, which suggests that the pathogen can easily survive the alkaline stress. Biofilm morphology at pH 7 is characterized by thick cell clusters, whereas at pH 10 it is characterised by thin layers of individual cells. These findings can help us understand how Salmonella enteritidis survives under highly alkaline conditions, potentially leading to the design of new and more effective disinfection strategies involving highly alkaline detergents.

The island of Malta has a rich heritage, which is evident in the rustic appeal of this island. Ġbejna forms an integral part of the Maltese food heritage. This artisan product is made from sheep or goat milk curds and aged for several months to develop its distinctive taste. During the ageing process, the cheese can become spoiled by fungi and unsafe for human consumption. This is a significant public health risk and a financial liability for producers. Conventional microbiology techniques do not detect these slow-growing pigment-less fungi early, allowing occasional distribution of contaminated products. We propose the use of hyperspectral imaging to detect these fungi during the early stages of cheese production. In contrast with a typical digital camera, which compiles the light signal into three broad wavelength bands; red, green and blue, a hyperspectral camera records numerous narrow and contiguous wavelength bands reflected from an object. This produces a series of images, each corresponding to the reflected electromagnetic energy in the respective narrow band of wavelengths. This image series may, in turn, be used for early fungal detection and identification. To test this hypothesis, a model cheeselet was produced to conduct compatibility and stability studies, through measurements of colony forming units, water activity, moisture levels, pH, protein and sugar content. The ġbejna model was then challenged with fungal strains isolated from commercial ġbejna and imaged using a hyperspectral camera. An algorithm is under development to differentiate contaminated samples from uncontaminated samples using image analysis and multivariate statistics.

Antibiotics are used extensively in agriculture as therapeutics, and in some countries, for prophylaxis and as growth promotors. Alarmingly, there is a potential resistance transmission pathway from animals to humans through food. We analysed the microbiome and resistome of sixteen broiler chickens, which are raised for meat production. DNA was extracted from caecal samples taken at days 27 and 34 posthatch from broilers, half of which had their diets supplemented with a mannan rich fraction. Paired end sequencing was performed using an Illumina HiSeq 4000. The data was analysed by MGnify to generate taxonomic read files. The microbiome was analysed using Calypso software and the antibiotic resistance genes (ARGs) identified using the ARGs-OAP pipeline. The main phyla detected in all samples was Firmicutes and Bacteroidetes. Clostridia was the main class detected and Clostridiales the main order. Faecalibacterium, Lactobacillus and Bacteroides were the main genus detected, which are common in the broiler caecal microbiome. There was an increase in Bifidobacterium in the treated group. Principle component analysis showed that both time points cluster together. Rarefaction analysis confirms that a sufficient sequencing depth was obtained. Tetracycline resistance comprised the greatest proportions of ARGs present, followed by the aminoglycoside, macrolide, vancomycin, beta-lactam and bacitracin classes. Further analysis of the sequences will allow for full characterisation of the resistome between treatment groups. The antibiotic resistance residues in food animals may have the potential to disseminate antibiotic resistance to the human community via the food chain.

Shiga-toxin producing Escherichia coli (STEC) is a foodborne zoonotic pathogen of significant public health concern. Ruminant animals are considered the primary reservoir of STEC. STEC predominantly colonises the lower gastro-intestinal tract, termed the recto-anal junction (RAJ). The number of STEC shed in the faeces of ruminants can vary widely with some animals, termed ‘super-shedders’ (>Log104 c.f.u. g−1 faeces), high risk carriers of the pathogen. The objective of this study was to sample a large cohort of Irish sheep, with quantitative and qualitative analysis of each sample for STEC. RAJ swab samples (N=410) were collected over a 9 month period from an ovine slaughtering facility. Each swab was enriched in 30 ml of modified Tryptone Soya Broth with Novobiocin at 41.5 °C for 5 h and subjected to a quantitative real-time PCR assay to detect and enumerate serogroups O157 and O26 in super-shedding animals. Incubation was allowed to continue for 24 h and shiga-toxin prevalence was assessed using a targeted qualitative real-time PCR assay. Eight O157 strains were isolated, of which six were super-shedding strains. The incidence of stx, O157 and O26 positive swabs was 49.3 %, 1.95 % and 0.24 % respectively. The prevalence of stx1, stx2 and stx1/stx2 virulence factors in isolated strains was 15.9 %, 8.8 % and 22.4 %. Additionally, the occurrence of stx1/stx2 in combination with eaeA in strains was found to be significant according to Pearson’s correlation and a paired T-test. In conclusion, these results underline the risk Irish sheep pose as a potential source of STEC infection.

Shiga toxin producing E. coli (STEC) is a foodborne pathogen which causes severe, debilitating, and sometimes fatal, illness. Ireland consistently has one of the highest incidence rates of human STEC infection in Europe. Cattle are one of the primary reservoirs for STEC, excreting the pathogen in their faeces. The amount of pathogen excreted varies greatly, with animals shedding >log104 c.f.u. g−1 faeces being termed ‘super-shedders’. Human infection can occur from faecal contamination of meat, dairy, fresh produce or drinking water. STEC can survive for extended periods in soil, slurry and water, although the exact means is unknown. The objective of this study was to examine phenotypic traits potentially relevant to extended environmental survival in two strain banks: (1) clinical and bovine STEC, in comparison with non-STEC, isolated from the production environment and (2) E. coli O157:H7 of known shedding status. The strain banks were assessed for biofilm-forming abilities and the ability to adhere to the muscle component collagen-I, using a 96-well crystal violet assay, where the absorbance of bound cells indicated biofilm formation levels or adherence to collagen. Extracellular components involved in attachment were assessed using Congo red agar and pellicle formation was also examined. Phenotypic traits potentially related to extended environmental persistence were observed more frequently in non-STEC. However, these traits were also observed in some STEC isolates, showing phenotype is strain dependent indicating a risk for enhanced environmental survival of some STEC isolates. The shedding status of E. coli O157:H7 is not dictated by the investigated characteristics alone.

Antimicrobial Resistance (AMR) occurs when micro-organisms develop the ability to counteract antimicrobial drugs through previous exposure. The past decade has seen an increased prevalence of AMR bacteria due to widespread antimicrobial use (AMU). A substantial share of antimicrobial consumption is attributed to animal production, particularly within the pig industry as it is recognised to be a high user of antimicrobials. Considerable research has focused on the scientific mechanisms of AMR; however, limited literature exists regarding the perceptions of food producers towards AMR. Four databases; Web of Science, PubMed, ScienceDirect and Google were used to search keywords; ‘UK,’ ‘pork,’ ‘supply’ and ‘chain’ to identify relevant papers. Each paper was inspected to ensure that a pork supply chain was illustrated. Interviews were conducted respectively with professionals working in the pork sector to verify the chain and uncover perceptions towards AMU and AMR. Results verified the accurate mapping of the pork chain, enabling professionals to highlight areas of AMU. Stakeholders perceived antimicrobials as useful for the treatment of diseases however, opinions varied regarding the transfer of AMR to humans and the effects this may have on health. To combat the problem of AMR and high use of antimicrobials within the pig industry, it is necessary to identify the key stages of AMU and to uncover stakeholder perceptions along the pork chain. Results will be verified using surveys and an intervention will be designed to enhance knowledge and understanding of AMR to influence a change in behaviour and thus, positively impact farmers on-farm practices.

Phytophthora are a genus of microbial, filamentous eukaryotes that morphologically resemble fungi but belong to the Oomycete class. Phytophthora species include some of the most destructive pathogens of plants, including many economically important crops and forest species. They represent one of the biggest threats to worldwide food security and natural ecosystems. Phytophthora are notorious for secreting large arsenals of effector proteins which facilitate infection by degrading host cell components, exploiting host nutrients, dampening host immune responses and inducing necrosis. Compared to other taxonomic groups, there is a paucity of OMICs data available to study Phytophthora species. To this end, we have used an LC-MS/MS strategy to perform the first large-scale profiling of the secretomes of three Phytophthora species that are an increasing threat to global forest ecosystems: Ph. chlamydospora, Ph. gonapodyides and Ph. pseudosyringae. Together, Ph. gonapodyides and Ph. chlamydospor are present the two most widespread Phytophthora species, having been found in a wide range of habitats globally. Ph. pseudosyringae has been identified as the cause of oak and beech decline across Europe and America. Here, we use mass spectrometry to characterise the secretome of these Phytophthora species by identifying proteins secreted into different growth media. We detect a number of important effector families including proteins involved in the breakdown of plant cell wall carbohydrates (CAZymes) and toxin families such as necrosis-inducing proteins. Our results provide important insights into understanding the molecular mechanisms of Phytophthora infection.

Dietary deficiencies are major cause of malnutrition in the developing world, particularly vitamins deficiencies such as riboflavin, which lead to various health disorders. Traditional plant fermentation and their indigenous starter cultures such as Bacillus subtilis might provide solutions to bioenrich riboflavin. We aimed to investigate this idea on the example of B. subtilis derived from iru an alkaline fermented condiment playing an important role in rural Nigeria. After initial isolation, identification and safety assessment, roseoflavin exposure was used to obtain riboflavin overproducing mutants. These were further analysed for functional characteristics. Bàcillus species’ (n- 123) were isolated from iru. Initial riboflavin production in supportive growth medium ranged from 50.3 to 479.0 µg l−1 for 27 out of 123 strains evaluated. Subsequent gràdual exposure to 200 mg l−1 roseoflavin increased riboflavin production in the three best producing strains from 350 µg l−1 to 542 µg l−1, 479 µg l−1 to 580 µg l−1 and 362 µg l−1 to 618 µg l−1. This increased riboflavin in lab-scàle iru fermentation by over 150 percent to 0.12–0.14 mg g−1 and near the recommended daily intake while retaining desired proteolytic and esterase activity. This research provides important proof of concept for the bioenrich the of traditional B. subtilis based plant fermentations used across sub-Saharan Africa and possibly other areas globally.

The eco- friendly improvement of crop yield is a mammoth task that must be tackled in order to meet the ever increasing world population in need of food. Plant growth promoting rhizobacteria (PGPR) are organisms known to increase the growth of plants, by directly or indirectly facilitating crop yield by a number of mechanisms. Vegetables are stable foods rich in numerous vitamins. Low income countries get a balance diet through regular consumption of vegetables like Amaranthus hybridus which is regularly consumed with highly starchy food across the Southern states of Nigeria. This study investigated the effect of different bacterial suspension samples in increasing the growth parameters of Amaranthus hybridus through different plant growth conditions and treatments, determination of rhizospheric and endophytic bacterial colonization, RNA extraction, cDNA synthesis and qRT – PCR analyses, and Microarray hybridization. Bacterization by microorganisms tagged ADK 1, ADK 2, ADK3, ADK4, ADK 5, ADK 6 and ADK 7. The pot trial showed an increase in the growth yield of Plant affected by ADK 5 and ADK 7. A synergistic effect of ADK 5 and ADK 7 did not give an increased yield as each individual effect. An Amaranthus hybridus transcriptome analysis revealed that several genes showed differential expression after inoculated by ADK 7. These genes are implicated in stress response and hormone pathways. Investigations into the bacterization of indigenous vegetables by indigenously isolated bacteria is an eco-friendly agricultural practice to be promoted.

Biofilms are sessile communities of microorganisms embedded within a self-generated extracellular polymeric matrix. Such biofilms are found for instance in adults with cystic fibrosis, with pulmonary infections with the Gram-negative bacterium Pseudomonas aeruginosa being particularly common. This infection in CF patients is commonly managed with antibiotic dry powder inhalers, one of which is the aminoglycoside tobramycin. The activity of tobramycin has been well characterized in vitro, but current models that have been used are not very representative for lung infections, and better models would provide a significant advantage as these could be used, for instance, to improve the formulation of dry powder inhalers. For instance, one question that has not been addressed with current models is whether the size of drug particles emitted from a dry powder inhaler influences the efficacy of the anti-biofilm activity of the antibiotic. In this project, we utilized the Next Generation Impactor (NGI), which is a pharmaceutical instrument used to separate particles into size fractions. We used the NGI to separate tobramycin particles into different sizes and tested the influence of these particles on eradication of P. aeruginosa biofilms, which were grown using as colony biofilms that closely mimics conditions in the lung where biofilms are grown on a substrate-air interface. Preliminary evidence indicated smaller tobramycin particles are better in eradication of P. aeruginosa biofilms as compared to larger particles. Our results may represent a step towards improving the formulation of tobramycin dry powder inhalers to be effective in eradicating P. aeruginosa biofilms.

Streptococcus pneumoniae is a common nasopharyngeal resident in healthy persons, but remains a major cause of pneumonia, bacteremia and otitis media despite vaccines and effective antibiotics. There is an urgent need for novel therapeutic approaches, but such advances require a detailed knowledge of S. pneumoniae biology and its shift from commensal to pathogen. To better understand pneumococcal biology and infections, we need sensitive in vivo imaging technologies. To this end, bioluminescence imaging can be used, for example, to evaluate anti-infectives, intraspecies interaction and pneumococcal virulence non-invasively. A click beetle luciferase (CBR-luc) containing vector pPP3 under the control of putative highly expressed pneumococcal promoters was constructed. The CBRluc providing red-shifted light production was integrated into known sites in the S. pneumoniae genome. The constructs were compared to a lux-based exist system expressing bacterial luciferase using in vitro growth experiments. The results revealed that CBRluc tagged bacteria, PphrA::luc-wt, showed robust activity of bioluminescence in exponential phase that is maintained during stationary phase, whereas, lux-expressing pneumococci emitted a light signal with high background that peaked during exponential phase and was significantly reduced in intensity during stationary phase. Initial findings demonstrate that the CBRluc reporter system is more efficient than lux, providing a potential platform for utilization in understanding of the mechanisms of pneumococcal pathogenesis in vivo system.