- Volume 2, Issue 7A, 2020

Background and objective: Salmonella is the underlying cause of foodborne diseases and poses a major public health problem worldwide. Research has developed a method to efficiently treat cancer using some of the same bacteria behind food poisoning, one of these bacteria is Salmonella which targets and penetrats tumours specifically by being attracted to the compounds produced by tumour cells and accumulating at the tumour site and inducing inflammation. In this project we aim to investigate the mechanism of Salmonella Typhimurium which has a tremendous ability to invade, replicate and compete to survive inside the cells by virtue of effector proteins such as: sipA, sipB, and AvrA which it possesses.

Method: The S.Typhimurium strains used are wild-type SL1344 ( ΔsipA, ΔsipB, ΔavrA and VV341) and attenuated strain of SL7207. Bacteria were cultured in LB broth in a 37 °C shaker overnight to reach a stationary phase before using it to infect B16F10 (mouse melanoma).

Results: The initial results show that the infection of B16F10 with wild-type SL1344 had a high level of invasion compared to the low number of bacteria with the deletion of sipB which impaired its entry into the cell. Similarly, the mutant strains ΔsipA and ΔavrA show an increasing number of intracellular bacteria, like the wild-type strain. We will be investigating further on the innate mechanisms of Salmonella in disrupting tumour growth and progression, that might help maximize the potential of using these bacteria in monotherapy or in tandem with other useful therapies.

Studies have implicated that Adherent-invasive Escherichia coli (AIEC) in the aetiology of Crohn’s Disease (CD), a chronic inflammatory bowel disease (IBD). CD-associated AIEC are characterised by an ability to adhere to and invade intestinal epithelial cells and replicate intracellularly in macrophages. This occurs as AIEC form vacuoles within phagolysosomes, preventing macrophage-mediated killing. However, little is known about the interaction of macrophage proteins with AIEC.

In this study, we used an in vitro infection model to identify macrophage proteins associated with AIEC intracellular replication. We identified the importance of proline-rich tyrosine kinase 2 (PYK2) in AIEC infection. PYK2 is widely expressed in epithelial cells and hematopoietic cells and plays essential roles in tumorigenesis and tumour progression. PYK2 is also overexpressed in patients with intestinal and colorectal cancer (CRC). CRC is one of the major long-term complications of IBD, especially in patients with long disease duration, a large extent of colitis, and uncontrolled inflammation. Moreover, PYK2 has been identified as an IBD risk loci via large scale genome-wide association studies.

Our in vitro models have demonstrated that the addition of PYK2 inhibitor PF-431396 during infection results in a significant decrease in intracellular replication of AIEC. This has been quantified using fluorescence immunostaining and imaging flow cytometry. These results suggest that PYK2 may play a role in intracellular bacteria survive and replication. Our future work aims to further understand the relationship between PYK2, AIEC and CD.

Foot mycoses are a frequent disease that represents a public health problem worldwide. This study aims to evaluate the epidemiology of foot mycoses among footballers in Owerri, Imo State, in order to determine the fungal etiological agents and to identify possible risk factors. To investigate the treatment and preventive measures for the susceptible groups. A total of 485 samples were collected; tinea unguium were confirmed in 88.2% of cases. A prospective study of fifty footballers was undertaken during one year (2018-2019). A complete mycological diagnosis was carried out on all footballers. The results obtained showed that out of the 50 toe nails samples of footballers examined, 28(56.0%) had positive cases of the infection on direct microscopy which served as the screening test. The causative pathogens of Onychomycosis isolated from fungal culture are dermatophytes , and the most frequent pathogen was Trichophyton rubrum 15(30.0%), yeast Candida albicans 9(18.0%). Non-dermatophyte molds were observed in 8(16.0%) cases and Fusarium sp. was the frequent genus 14(28.0%)and Aspergillus sp. 4(8.0%). The main predisposing factors of fungal foot infections were practicing ritual washing (56.6%) and frequentation of communal showers (50.5%). Confirmatory test such as germ-tube test was done on the Candida albicans isolate for proper identification. The age group 31- 35 years had the highest prevalence of onychomycosis 12(92.31%) and age group 16-20 years presented the lowest prevalence of onychomycosis 4(28.57%). Proper care of toe nails and boots is necessary to prevent the increasing rate of this infection.

In biological science, metagenomics has revolutionised the process of drug discovery. This is because metagenomics serves as a tool for in depth characterisation by examining, sequencing, replicating and identifying the whole DNA/genome collected from any mixed population (Mahapatra et al., 2019). Metagenomics is used to study Carbohydrate Active Enzymes (CAZymes) containing microbial communities due to molecular-based culture-independent methods (Kunath et al., 2017). CAZymes are made of multiple domains; each domain serves as the key mediator for a specific function for the protein and structure. In the Carbohydrate-Active enZYmes (CAZy) database 20% of all protein domains are identified as domains of unknown function (DUFs). DUFs are mostly ignored due to not being the most abundant in many genomes. However, in a paper by Goodacre 2014, DUFs are shown to be essential and likely related to the organism survival function due to their relation to essential proteins(Goodacre et al., 2014). In the CAZy database multiple DUFs are associated with catalytic CAZyme domains, which could result in them having a similar function or increase their catalytic activity. We present DUFs that are considered to have the capability to help CAZymes break down polysaccharide chains and tools/methods we used to achieve these results.

Non-antibiotic compounds including metals and biocides co-select for clinically relevant antimicrobial resistance (AMR) genes. Presently, there is little research looking at the effects of plant protection products (PPPS) such as herbicides and insecticides on the development and spread of resistance. Agricultural activities require the direct application of PPPs in large quantities and at high concentrations to soil. These chemicals are applied alongside antibiotics and manures which may contain antibiotic residues, other selective agents and resistant bacteria. The selection pressure exerted by this mixture of chemicals may result in enrichment of resistant bacteria and/or the exchange of resistance genes between environmental and clinically relevant bacteria. The impact of these chemicals on AMR and microbial diversity in terrestrial environments is poorly understood.

PPPs from different substance groups were tested for antimicrobial activity using the SELECT method developed by Murray et al. (2019; under review). In this method, bacterial communities were exposed to PPPs and selective concentrations were identified by a significant reduction in community growth. Results from these experiments determined the concentration at which long term evolution experiments were carried out in soil microcosms. Selection for AMR was investigated using metagenomic analysis and targeted real-time PCR.

Many bacterial genes encode proteins that are secreted extracellularly. These proteins can be considered cooperative because all surrounding cells can benefit from their production. Therefore, it has been hypothesized that these cooperative genes would more frequently lie on mobile elements, such as plasmids, which can transfer to other cells. This could stabilise cooperation, leading to the prediction that plasmids should carry proportionally more cooperative genes than the less mobile chromosome. However, it is unknown whether this prediction holds across the bacterial tree of life. To address this, we analysed the gene content of the chromosome and plasmid(s) of 1620 genomes comprising 51 diverse bacterial species. We find that across species analysed, plasmids do not carry proportionally more cooperative genes than the chromosome. Contrary to prediction, the role of mobile elements in promoting cooperative behaviour is highly variable across bacterial species.

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 uptaketo 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.

Phosphate is added to drinking water by UK water companies as a treatment to prevent the corrosion and metal leaching, like lead, in pipes. However, phosphate is a nutrient for microorganisms, and it can favour biofilm formation in Drinking Water Distribution Systems (DWDS), which can alter the water quality and safety. This study analyses the effect of phosphate addition on biofilm formation over different materials and its consequences for drinking water quality by i) using controlled experimental pipeline facility representative of a real-scale DWDS with high-density polyethylene coupons and ii) using a small-scale DWDS biofilm reactors with lead coupons. Biofilms developed over one month were exposed to the effect of different phosphate dosing and compare with UK normal water phosphate concentrations. During the experiment, physico-chemical analysis of water and microbial analysis of biofilms was carried out. Sequencing analysis of the 16s rRNA gene, from extracted DNA obtained from biofilms, provided information on any bacterial changes, and Scanning Electron Microscopy gave information about the biofilm organization. The results indicate that microorganisms find more difficult to establish and develop biofilms under high phosphate dosing, resulting in biofilms with less cells. Also, some physico-chemical parameter seems to be affected by phosphate dosing, like chlorine and lead. It is expected that differences in the biofilm community will be found depending on phosphate dosing. This study will provide information on the effect of phosphate on biofilm development in different pipes materials, which will facilitate to adjust an optimal phosphate dose to prevent plumbosolvency in DWDS.

Antimicrobial resistance is one of the greatest challenges for humanity. Patients, especially, admitted to the intensive care unit have been exposed to higher risk of healthcare associated infections mostly caused by antibiotic resistance since the imprudent use of antibiotics over the years. The discovery and development of new antibiotics are facing difficulties with the continuous evolution of drug resistance in bacteria, and the reduced investment in R&D for antibiotics from pharmaceutical companies. Novel strategies are urgently needed to control this pandemic threat of antibiotic resistance. Antibiotic combination with two or more drugs may be useful in targeting resistant gram-negative bacteria by producing synergistic effect and enhanced bactericidal activates. In this study, we determined the combination of rifampicin and colistin against Extended Spectrum Beta Lactamase (ESBL), carbapenemase producing and colistin resistant Enterobacteriaceae using chequerboard method and time kill curves. We measured the combination effects based on Fractional Inhibitory Concentration index (FICI) and the efficacy of bacterial reduction comparing to that of single antibiotic. Interestingly, we found that the combination of rifampicin and colistin showed synergistic activities against the tested bacteria, indicating FIC index ≤0.5. The time kill curve shows that the two drugs combination exhibits 99% kill whilst the single antibiotic had no activities. Thus, the combination of rifampicin and colistin demonstrated synergistic activity with reduced MIC and the increased rate of killing against both ESBL and carbapenemsase producing and colistin resistant Enterobacteriaceae.

Global warming has changed the range of tick species and the tick borne diseases (TBDs) they carry; there is an increasing need to assess the risk and impact of TBDs. This study focused on TBDs of Dogs in Nigeria, where despite a large burden of ticks and TBDs there is little data on the prevalence of either for veterinarians and human health professionals to make informed decisions regarding treatment and control.

Most existing studies on cattle have indicated that Anaplasma, Babesia, Theileria, Ehrlichia, Hepatozoon and Candidatus Neoehrlichia species are present in Nigeria. However, dogs kept in much closer contact with their owners, may represent a higher risk of zoonotic TBDs transmission. This study used a combination of morphological and molecular identification of ticks and tick borne pathogens, and IDEXX 4Dx kits for pathogen antibody detection from canine blood, we examined 93 dogs to date.

Rhipicephalus sanguineus was the most abundant tick found on Nigerian dogs. Babesia species were the most abundant tick borne pathogen, followed by Ehrlichia species. This study serves as the first report of Babesia microti like species, Theileria parva, Babesia bovis, Anaplasma phagocytophilium and Ehrlichia chaffeensis in Nigerian dogs. There was no evidence of Borrelia Burgdorferi sensu lato nor Dirofilaria species in Nigerian dogs. The presence of Babesia microti and Anaplasma phagocytophilium is a direct public threat. Further testing of additional 200 dog samples and multivariate modelling of demographic risk factors, will enable us develop treatment and control guidelines for TBDs for Nigerian Veterinarians and Dog owners.

A recent study screening over 1000 drugs has shown that non-antibiotic drugs (NADs), including human targeted drugs, have been shown to have antimicrobial effects on representative strains of human gut bacteria. NADs are present in both wastewater effluent and freshwater systems, where they may only be partially metabolised. Concentrations of antibiotics found in the environment have been shown to select for resistance in complex microbial communities, and it is thought that concentrations of NADs in the environment may also select for resistance. This project aims to determine if NADs select for antimicrobial resistance, by investigating effects in a complex bacterial community. Pharmaceuticals from 7 different drug classes have been screened for antimicrobial activity. The most potent compounds will be used in exposure experiments to identify any existing antimicrobial resistance genes that confer cross-resistance to both antibiotics and NADs using a metagenomics approach. In addition, changes in community composition will be investigated to determine if there is enrichment for opportunistic pathogens after exposure to NADs. This project ultimately aims to inform on environmental water quality standards and environmental risk assessment of wastewater treatment effluent in order to mitigate the growing problem of environmental antimicrobial resistance.

Background: Multidrug-resistant efflux pump genes oqxAB-encoding plasmids are wildly disseminated in livestock, and also identified in human samples. Giving the high presence in environment and multidrug-resistant nature of oqxAB, deeper understanding of oqxAB-encoding plasmids is necessary.

Objective: To investigate the prevalence, virulence, and phylogenetic of oqxAB-encoding plasmids.

Method: A total of 43 oqxAB carrying Escherichia coli were isolated from human and livestock from China and Hong Kong, these isolates were characterised by disc-diffusion susceptibility test and PCR. The 43 samples were sequenced using PacBio or Illumina platforms, and analysied with five oqxAB-encoding plasmid sequences extracted from Genbank. in vivo pathogenicity study performed by injecting E. coli J53 transconjugants containing oqxAB plasmid into Galleria mellonella larvae.

Result: Among the 48 oqxAB-encoding plasmids, 27 were IncFII F18 plasmids, 12 F16 plasmids, six F33 plasmids, two F24 plasmids, and one F14 plasmid. Virulence regions in plasmids with same Inc group encoded similar set of virulence factors and antibiotic resistance genes. Co-existence of blaCTX-M, fosA3, and oqxAB genes was identified in all F33 plasmids and one F18 plasmid. in vivo pathogenicity study suggested oqxAB plasmids caused significant health deterioration in larvae but no significant increase in death.

Conclusion: Our data shows similar sets of pathogenic factors are frequently co-carried by oqxAB plasmids of same Inc group, suggesting high correlation between plasmids identified from China and Hong Kong. Co-existence of fosA3, blaCTX-M, and oqxAB suggests resistance to most clinical antibiotics. in vivo test indicates oqxAB plasmid can increase pathogenicity of the host bacteria.

Background: Acinetobacter baumannii (AB) was declared an antibiotic-resistant “Priority 1 pathogen” by WHO. We sought to investigate the predisposing risk factors to this pathogen.

Methods: In a retrospective study, adults who were admitted to Sohar hospital during 2016-2017 and had a positive laboratory-confirmed culture of AB were studied.We classified patients into 2 groups based on 30-day, all-cause mortality and compared the characteristics. Exploratory classification and regression tree (CART) analysis was performed to explore risk factors for mortality to include to a logistic regression model.

Results: A total of 321 patients were included, age was (Mean±SD) 57.42±20.22, male gender was 180(56.07%), mortality was 140(44%). Survivors vs deceased had; length of stay 38.25±88.74 vs 51.31±79.19 (p=0.002),multi-drug resistantisolates 134(51.34%) vs 127(48.66%) p=<0.001, critical care admission 35(38.04%) vs 57(61.96%) p=<0.001, comorbidities 114(47.50%) vs 126(52.50%) p=<0.001 and history of invasive procedures 82(59.85%) vs 55(40.15%) p=0.27. Logistic regression revealed that the odds of dying increase by a factor of 1.044 for every additional year of age, 1.844 times higher for male compared to female, 4.412 times higher for patients admitted into critical care units compared to general wards, 3.138 times higher for patients admitted with a diagnosis of infection, 2.356 times higher for patients with hospital-acquired AB infection compared to community-acquired.

Conclusion: Both modifiable and non-modifiable risk factors are associated with mortality and overall health status may contribute to infection outcome. Stabilization of comorbidities and effective antimicrobial treatment could be the mainstay of successful prevention.

The World Health Organization considers carbapenem-resistant Enterobacteriaceae (CRE) an urgent public health threat due to global prevalence, limited treatment options, and high mortality rates. Carbapenem-resistant Klebsiella pneumoniae (CRKP), the most common CRE species, is especially prevalent in long-term acute care hospitals (LTACHs) in the United States. Among patients colonized with CRKP, only a subset develop clinical infection. We used CRKP whole-genome sequences and associated clinical metadata from unique patient samples from 20 LTACHs (n=366) to (1) identify both clinical and microbial features associated with infection compared to colonization, and (2) evaluate the contribution of microbial features in infection prediction. Modifiable clinical features associated with infection could inform clinical practice, while microbial features could help predict clinical infection. We performed L2 regularized logistic regression 100 times for each feature set. Clinical predictors of infection include having a central line or gastrostomy tube. Genomic predictors of infection include iron scavenging genes (known to be associated with invasive infections in healthy hosts) and the number of antibiotic resistance genes. Furthermore, we found that clinical and genomic features (separate or combined) have similar predictive power (AUROC IQRs: clinical=0.56-0.64, genomic=0.54-0.63, combined=0.59-0.67). This suggests that genomic features may be associated with certain clinical features. These results provide insight into potential clinical and microbial drivers of CRKP infection in LTACH patients and provide a starting point for investigating the biological basis of infection, identifying patients at high risk of infection, and devising targeted strategies to prevent infection.

Pseudomonas aeruginosa and Staphylococcus aureus are bacteria pathogens that cause a myriad of infections affecting various sites in the body including the eyes, ears, lungs, skin, heart, bones, and blood amongst others. These bacteria can be disseminated via the blood to other parts of the body away from the primary site of infection and consequences vary from mild to severe with death occurring in certain instances. Both bacterial infections can occur individually, as well as in co-infection resulting in even worse outcomes. P. aeruginosa and S. aureus exhibit multidrug resistance against current antibiotic treatment regimens, which accentuates the challenge in managing the infections caused by these bacteria. To prevent the looming era of untreatable bacterial infections, alternative treatment regimens that are cost effective and accessible are needed. To explore novel treatment options, twenty-five organic compounds comprising fatty acids and their derivatives were screened for antibacterial activity in broth microdilution assay to determine the minimum inhibitory concentration and minimum bactericidal concentration against both P. aeruginosa and S. aureus. Five candidates (N–nonanoic acid, butyric acid, heptanoic acid, palmitoleic acid, and isopropyl myristate) were effective against P. aeruginosa. Seven candidates (N–nonanoic acid, palmitoleic acid, tridecanoic acid, sebaic acid, undecanoic acid, monolaurin, and monocaprin) were effective against S. aureus. Candidates such as N–nonanoic acid and palmitoleic acid were effective against both P. aeruginosa and S. aureus, demonstrating that the same fatty acids show potential to be used against both Gram negative and Gram positive bacterial infections.

Hospital sinks in the UK have recently been under scrutiny as possible reservoirs for Gram-negative bacteria, especially carbapenem resistant Enterobacterales (CRE). These strains have been found in intensive care wards across the country and can re-enter the clinical environment, representing a risk to vulnerable patients.

Two sink waste traps known to be colonized with CRE were collected from a hospital and fitted to a vertical-draining and rear-draining handwash sink installed within a laboratory model sink system. Sinks were automatically flushed four times a day and, as per usual in the model, TSB was provided once daily to maintain microbial populations. Gram-negative bacteria were regularly monitored using selective culture, MALDI-TOF and antibiotic disk diffusion. The short-term effect of adding simulated IV fluids (5% glucose or 0.9% NaCl) and the impact of sink design on Gram-negative proliferation were investigated. Communities included Enterobacter asburiae; Klebsiella oxytoca; Pseudomonas aeruginosa and Citrobacter freundii, among others, including CRE. The addition of simulated IV fluids did not induce Gram-negative bacterial proliferation in the time frame of the experiment. Differences were observed in the fluctuation of Gram-negative levels after flushing between the different sink designs. Gram-negative numbers in vertical-draining sinks decreased immediately after the tap was flushed and subsequently increased between flushes. However, in rear-draining sinks, little fluctuation was observed. Hospital sink waste traps can harbour Gram-negative bacteria resistant to antibiotics. In our experimental conditions, the type of sink was the determining factor in the magnitude of fluctuation in Gram-negative populations while simulated IV fluids had little effect.

Aims: To isolate and characterize the bacteriocin activity identified by the genomic analysis of a novel enterococcal isolate, Enterococcus faecium W1

Methods and Results: Culture fermentates of a novel E. faecium isolate, W1 showed high levels of antimicrobial activity against Listeria monocytogenesisand a variety of other Gram+ve and Gram-ve bacteria. Ammonium sulphate precipitation of the fermentates followed by hydrophobic interaction chromatography showed bactericidal activity correlated with fractions containing only low molecular weight proteins. The same fractions had activity on human cancer cell line HT-29 measured by cytotoxicity assay (MTT) and apoptosis induction. Whole-genome sequencing of E. faecium W1 revealed five bacteriocin related gene clusters, a lack of virulence factors and lack of antibiotic resistance genes. To confirm that antimicrobial activity was correlated with the bacteriocin genes identified, selected bacteriocin genes were cloned and expressed as His-tagged proteins in E. coli. Bactericidal activity against L. monocytogenes was associated with protein purified by metal affinity chromatography. A structure-function analysis to investigate the role of key residues in activity is ongoing.

Conclusion: E. faecium W1 is a promising candidate for use as a probiotic supplement or in food preservation. The bacteriocin related genes are functional and could be improved for future use as antimicrobial and cancer cell therapeutics. Significance and Impact of the Study: There is a need for new approaches to the growing problem of antibiotic resistance. Expression of bacteriocins identified in E. faecium W1 could contribute to this endeavour in addition to its use as a probiotic organism.

The picornavirus foot-and-mouth disease virus (FMDV) is responsible for one of the most significant diseases of livestock, leading to large economic losses due to reduced productivity and trade embargoes for areas not certified as disease-free. The picornavirus non-structural protein 3A is involved in replication of the viral RNA genome and is implicated in host tropism of several picornaviruses. Deletions in the C-terminus of 3A have been observed in FMDV outbreaks specific for swine and such viruses are non-pathogenic in cattle. The mechanism for species specific attenuation of FMDV is unknown.

We have shown that FMDV containing a C-terminal deletion in 3A is attenuated in bovine cell culture and that the attenuated phenotype can be reversed by the JAK1/2 inhibitor Ruxolitinib (Rux), identifying a role for the induction of interferon stimulated genes (ISGs) in the restricted bovine tropism of the 3A-deleted virus.

Equine herpesviruses (EHVs) are enveloped DNA viruses infecting mainly members of the Equidae family and also members of other taxa. EHVs primarily causing respiratory disease, however EHV type 1 (EHV-1) can produce cases of a neurological disease, abortion and neonatal death, sometimes as regional outbreaks. Thus these viruses represent a welfare issue for the equine industry and scientific focus for researchers. EHV-1 presents a complex array of 12 glycoproteins on its surface envelope, but it is unclear which ones are important for virus cell entry and the role of each in host immune response. In order to investigate the contribution of these glycoproteins, pseudotype viruses (PVs) could provide a perfect study tool. In 2016, Rogalin & Heldwein successfully generated the first functional herpesvirus pseudotype, bearing the four glycoproteins gB, gD, gH and gL from human Herpes simplex 1. Our study is the first to attempt pseudotyping of EHV-1. We have employed homologous glycoproteins of EHV-1 in lentivirus PV generation, using different mammalian cells (e.g. epithelial, dermal, CNS) as transduction targets. The glycoprotein sequences obtained from an EHV-1 strain isolated from organs of aborted foetus during a significant outbreak in Normandy (France) in 2010. Future work will focus on the development of a PV assay for detection of neutralising antibodies in naturally infected horses for diagnostics and for vaccine evaluation.