- Volume 1, Issue 1A, 2019

Non-tuberculous mycobacterial pulmonary disease (NTM-PD) has rapidly increased in global prevalence over the last two decades. NTM-PD occurs mainly in patients with pre-existing structural lung disease and current treatment strategies are often ineffective and poorly tolerated. Outside of the cystic fibrosis population, the most common NTM isolates are from Mycobacterium avium complex (MAC). Mesenchymal stromal cells (MSCs) have potent antimicrobial and immunomodulatory properties including direct microbial killing and enhancement of phagocytic function. Their effect on MAC species is unknown. Human MSCs were infected with M. avium at a multiplicity of infection (MOI) of 2. Human monocyte-derived macrophages (MDMs) were also infected with a clinical isolate of M. avium at MOI of 2. After 4 h, MSCs were added at a ratio of 1 MSC:3 MDMs. After 24 and 74 h, colony counts were performed on supernatants and cell lysates. MSCs reduced total bacterial counts of M. avium by 24 % at 24 h (from 295×103/ml to 225×103/ml, P<0.05) and 40 % at 72 h (from 403×103/ml to 243×103/ml, P<0.05). MSCs reduced total bacterial counts of M. avium in infected MDMs by >40 % (from 381×103/ml to 209×103/ml, P<0.05) after 24 h and >70 % after 72 h (from 1050×103/ml to 314×103/ml, P<0.05). MSCs have modest direct antimicrobial effect against MAC, but potently enhance their killing by macrophages. Mechanistic studies are required to understand the mechanisms of the antimicrobial effect, with the aim of exploiting these therapeutically in pulmonary MAC disease.

Clostridium difficile is a Gram-positive spore-forming bacterium. Gut colonization is associated with a wide spectrum of gastrointestinal diseases, ranging from mild to severe, acute to persistent. Bacterial survival and multiplication are essential processes dependent in the maintenance of homeostasis, which is, in turn, inherently dependent on the integrity of the cell wall. Bacteria present different mechanisms to maintain cell wall integrity, particularly, to protect it from direct contact with the external environment, such as phosphate polymers, capsule, outer membrane and S-layer. The S-layer is an evolutionary conserved macromolecule present in almost all bacterial types, however its role(s) remain(s) to be clarified. The S-layer is ubiquitous in C. difficile strains and is comprised of a conserved and a variable region that confers strain specificity and faces the external environment. In C. difficile FM2.5 strain, the absence of S-layer resulted in impaired pathogenicity. Gut colonization of FM2.5 infected mice was significantly lower than mice infected with the wild-type strain R20291. In addition, FM2.5 showed compromised toxin activity and in vitromotility. Accordingly, FM2.5 failed to cause disease in mice and trigger a strong inflammatory response, contrary to mice infected with R20291. Reversion of the S-layer gene restored motility, toxin activity and the abilities to colonize and cause disease. It appears that the S-layer plays a crucial role in various bacterial processes, hence FM2.5 loss of virulence may be due both to the absence of S-layer at the cell surface and its role in other processes that aid to bacterial virulence.

The Enterobacter cloacae complex (Ecc) is a group of enteric Gram-negative bacteria that also exist as commensals in nature. Ecc bacteria are also responsible for nosocomial outbreaks, targeting primarily immunocompromised patients and causing a wide range of systemic infections. Infection is often fatal as management is complicated by the high-level multidrug resistance expressed by Ecc isolates. Indeed, the Enterobacter species represent the last ‘E’ of the ESKAPE pathogens, which are the global leading causes of nosocomial infections. Despite the clinical relevance of Ecc, little is known about their virulence-associated properties and pathogenicity. Bridging this gap in knowledge is fundamental to provide a blueprint to better tackle Ecc infections. The aim of this study is to explore how E. cloacae interact with human macrophages and identify the bacterial and host cell factors involved in this process. Differentiated, human THP-1 monocytic macrophages were infected with fluorescent E. cloacae for various lengths of time. Macrophage infection was analysed by confocal microscopy and images processed on ImageJ and LAS X. Data suggests that approximately 90 % of intracellular E. cloacae are killed by 5 h post-infection, but the remaining 10 % of the bacterial population survives and persists up to 48 h post-infection. We have also observed E. cloacae colocalising with early and late endosomes as well as lysosomes. Our current results suggest that E. cloacae can subvert normal phagocytic trafficking and possibly adapt to survive inside the acidic lysosomal environment.

Bovine tuberculosis is among the primary zoonotic disease caused by Mycobacterium bovis. A cross-sectional study was conductedon 315 cattle in selected areas of eastern Ethiopia, aiming to estimate the occurrence of bovine tuberculosis using comparative intradermal tuberculin skin test and assess cattle owners’ awareness on its public health implication. Random sampling method was applied in order to select animals from farm/household. Forty three farm/household owners of tuberculin tested animals were interviewed using pre-tested structured questionnaires. The overall prevalence of bovine tuberculosis was 20.3 % (n=64) in dairy cattle at recommended cut off >4 mm. From a total of 43 farms/households tested, 22 were positive; each farm exhibited at least one tuberculin positive reactor animal with a total herd level prevalence of 51.2 %. The prevalence of bovine tuberculosis in individual animal level was significantly different (χ2=45.2; P-value <0.001) in different sites. Farming system, herd size and other risk factors were significantly (P<0.05) associated with bovine tuberculosis occurrence. Of the total interviewed farm owners, only 33 % had the knowledge of or had heard about bovine tuberculosis and 23 % respondents were aware of the zoonotic importance of the disease. More than 50 % of the interviewees had shown their preference of raw milk consumption. The study showed bovine tuberculosis is highly prevalent. The majority of cattle owners lack awareness about the disease and its public health significance. Awareness rising about the disease, its transmission and zoonotic implication is of great importance for reduction and control measures.

Staphylococcus aureus is responsible for a variety of infections in humans and animals that particularly causes staphylococcal food poisoning when it present in foods. This study was aimed to isolate Staphylococcus aureus present on the shell surfaces and in the contents of chicken eggs, and determine antimicrobial susceptibility patterns. A total of 335 egg samples were obtained from open market (n=174) and poultry farm (n=161). A sterile cotton swab was used to sample the surface of eggs. After sterilizing the shells, the egg contents were sampled. Isolation of Staphylococcus aureus was done based on ISO. The isolates were subjected to antimicrobial susceptibility testing using disc diffusion method. Out of the total 335 eggs sample examined, 93 (27.8 %) samples yielded S. aureus. Out of these, 28 (17.4 %) were from poultry farm while 65 (37.4 %) were obtained from open market. Similarly, 63 (18.8 %) were from the shell while 30 (8.9 %) were from the content. The level of S. aureus in egg contents was significantly higher in the open market (CI=0.0962–0.6085; P=0.003). All 76 S. aureus isolates were resistant to at least one of the antimicrobials tested with overall 3.9–92.0 % level of resistance pattern showing higher resistant to penicillin (92 %), and ampicillin (89.5 %). Multiple drug resistance was detected in 86.8 % of the total S. aureus isolates. The study showed high level of S. aureus with considerable antimicrobial resistant pattern. Further study is needed to better define bacterial resistance to antimicrobial agents with emphasis of multiple drug resistant.

Salmonella Enteritidis is an important cause of human salmonellosis and food-poisoning associated with consumption of contaminated chicken eggs and poultry products. Objective of the study was to develop a serological diagnostic testfor the recognition of different Salmonella serovars in chickens. Here, phage peptide libraries were screened against 9 IgY samples from chickens infected with S. Enteritidis and 9 IgY samples with S. Hadar and the individual peptide binders were then identified using NGS. Twenty-nine peptides were identifiedin silico assessmentas being enriched specifically against IgY from multiple chickens infected with S. Enteritidis compared to those infected with S. Hadar. Twenty Nine peptides identifiedin silico assessmentwere thentested by both training and test cohorts of chicken IgY samples in ELISAs. The training set of samples was made upof IgY from 9 chickens infected with S. Enteritidis and 9 infected with S. Hadar. Seventeen peptides were selected as the most recognized specific peptides against S. Enteritidis infection and were then used against IgY samples from10 birds infected with S. Enteritids and20 birds with S. Typhimurium as a test cohort. Overall, for both training and test cohorts the peptide ELISA assay sensitivity and specificity were 90 % for detecting infections. The most discriminatory peptides by ELISA test were AEGEFEPQSARPS and AEGEFFVNRALINQ. The data demonstrated that the NGPD method could identify peptides that represented serovar-specific epitopes/mimotopes, these peptide have potentially important applications for the development of peptide based immuno-diagnostic assays for the recognition of Salmonella Enteritidis in chickens.

The genus Corynebacterium includes three potentially toxigenic species, C. diphtheriae, C. ulcerans and C. pseudotuberculosis, capable of causing diphtheria, a severe disease in humans. The aims of this study were to undertake Multi-Locus Sequence Typing (MLST) on a panel of both toxigenic and non-toxigenic C. diphtheriae and C. ulcerans strains (20 toxigenic and29 non-toxigenic C. diphtheriae, 17 toxigenic and 14 non-toxigenic C. ulcerans) isolated in the UK between 2004 and 2017, and use these results to determine the molecular epidemiology within the UK. Antibiotic sensitivity testing was also undertaken (20 toxigenic and 88 non-toxigenic C. diphtheriae, 17 toxigenic and 14 non-toxigenic C. ulcerans) and their profiles compared. The MLST results showed that C. diphtheriae and C. ulcerans isolates formed two distinct genetic populations and that C. diphtheriae isolates with intermediate penicillin resistance demonstrated sequence types which were genetically related. The results also showed that ST32 was most prevalent (31%, 9/29 isolates) amongst non-toxigenic C. diphtheriae. Non-toxigenic C. ulcerans isolates demonstrating intermediate penicillin resistance formed distinct genetic populations and appeared distantly related or unrelated. There were 75 % (15 isolates) of toxigenic C. diphtheriae isolates, 35 % (6 isolates) of toxigenic C. ulcerans isolates, 30 % (26 isolates) non-toxigenic C. diphtheriae and 43 % (6 isolates) of non-toxigenic C. ulcerans which demonstrated intermediate penicillin resistance. Linezolid and vancomycin were the only antibiotics which demonstrated 100 % sensitive profiles for all isolate groups. These data will help inform public health guidance and management of corynebacteria infections caused by these species.

Staphylococcus aureus (S. aureus) acts as a commensal in the microbiome of the skin and nasopharynx. However, on gaining access to the bloodstream it can cause an array of pathogenic outcomes. S. aureus can crowdsource the microflora to assist in becoming an opportunistic pathogen as our lab has recently published findings that co-inoculation of S. aureus with commensals, acting as pro-infectious agents, leads to a much more robust, virulent infection. This benefits S. aureus at doses where it would otherwise be cleared by the immune system. Pro-infectious agents do not need to be live commensals as isolated cell wall peptidoglycan also augments infection. This work aimed to assess the effects of inoculation with pro-infectious agents before and after infection with S. aureus. It was found that pro-infectious agents needed to be co-administered in order to fully augment infection. This gives mechanistic insight where S. aureus and the pro-infectious agents need to be in the same local environment or phagocyte to augment infection.

Streptococcus mutans is a pre-dominant bacterial species found in oral biofilms and participates in the production of dental caries via the generation of organic acids. The production of these acids results from the fermentation of carbohydrates present in a sugar-laden diet. As the acidity of an oral biofilm decreases, the demineralisation of the enamel of a tooth increases; leading to the formation of dental caries. To detect and measure the pH change occurring following a sugar challenge, ratiometric, fluorescent, pH-sensitive nanosensors were incorporated into oral biofilms. Confocal laser scanning microscopy revealed that the addition of glucose (1 % w/v) to an S. mutans biofilm resulted in a gradual reduction in the fluorescence intensity ratio during a 30 min period. This reduction in the fluorescence intensity ratio indicated a reduction in pH of the biofilm over time as the glucose was being fermented, resulting in the production and secretion of acids into the extracellular matrix of the biofilm. Additionally, a reduction in pH was detected – using widefield microscopy – in starved, planktonic S. mutans when treated with glucose. Over the course of 30 min, the pH of the medium was reduced from pH 5.3 to pH 3.3 as the glucose was fermented by the bacteria. These findings will help us map pH changes in oral biofilms as we examine potential methods of preventing the acidification of oral biofilms and the eventual demineralisation of the enamel; leading to the reduction in dental caries and an improvement in the standard of living of those effected.

Extraintestinal pathogenic Escherichia coli strain CFT073 is a prototypic urosepsis isolate. CFT073 is of serotype O6:K2:H1 and thus has an O-antigen. The wzy gene encodes the O-antigen polymerase Wzy, which catalyses the polymerisation of O subunits into a long chain polysaccharide. A CFT073 Δwzy mutant was constructed using the λ Red recombination system. The lack of O-antigen was confirmed by LPS purification and staining with Pro-Q Emerald 300. CFT073 Δwzy and wild-type CFT073 were tested for their bacteriophage sensitivity. ΦEB49, a member of the Myoviridae, can mediate generalised transduction in strain CFT073. CFT073 Δwzy and wild-type CFT073 mixed with the same concentrations of ΦEB49 phage were compared. Confluent growth was evident on all CFT073 Δwzy plates, whilst lysis was apparent on all CFT073 plates. The inability of ΦEB49 to lyse CFT073 Δwzy suggested that this phage binds to O antigen, hence the decreased O antigen of CFT073 Δwzy, compared to wild-type CFT073, prevented phage lysis from occurring. These data indicate that CFT073 Δwzy is not a susceptible host for ΦEB49.

Neonatal meningitis Escherichia coli (NMEC) is the leading cause of gram-negative meningitis in neonates, and it contributes to neonatal morbidity and mortality globally. The prototypic strain of NMEC, E. coli RS218 possesses the K1 capsule and has been widely employed in the study of NMEC pathogenesis. Previously, our laboratory has utilised relatively large volumes of culture to assay serum bactericidal activities, to garner valuable insights into bacterial immune evasion strategies. However, these methods can be labour intensive, time consuming and are not easily adaptable for high throughput applications. To overcome these limitations, a smaller volume real-time assay was developed. Bacteria were cultured in 100 µl volumes and were sub-inoculated for logarithmic growth. A slow kinetic absorbance assay was established on the Optima Fluorostar microplate reader, which enabled the accurate and reliable measurement of bacterial growth. Subsequently, bioluminescence was incorporated into the assay to facilitate the measurement of bacterial viability in real-time. Bacteria were rendered bioluminescent via electroporation of the pIlux plasmid or alternatively by the addition of exogenous beetle luciferin and recombinant firefly luciferase. The utility of these approaches in the determination of bacterial complement evasion is reported below.

Recently, we have demonstrated that the pathogenesis of Streptococcus pneumoniae bacteraemia contains a concomitant phase of intracellular replication within splenic macrophages in mice (Ercoli et al. Nat Micro. 2018). In the present study, we aimed to determine if intracellular replication of pneumococci may play a role in the pathogenesis of sepsis in humans, using two innovative approaches. We used a model (Chung et al. ALTREX.2018) involving ex vivo perfusion of human spleens from elective splenectomy patients (REC reference: 18/EM/0057). Organs were infected with 6.5×107 c.f.u. of pneumococci, and serial biopsies and ‘blood’ samples were taken at predetermined times. Samples were analysed by colony counts, confocal microscopy and flow cytometry. Additionally, infected tissue samples were taken for preparation of organotypic slice culture time courses. Bacteria injected into the perfusion circuit were rapidly cleared at early time points post-infection, recapitulating what is observed in experimental murine sepsis. Bacterial counts in the spleen increased, providing initial evidence of intracellular bacterial persistence. Microscopy analysis indicated that bacteria could be localised to splenic macrophages, with the size of infectious foci increased over time. Z-stack microscopy localised bacteria within cell membranes, indicating the infection was predominantly intracellular. In ex vivo slice cultures increasingly large numbers of pneumococci were cultured over time, further indicating intracellular replication. In conclusion, we provide evidence for a role of intracellular replication of pneumococci in human splenic macrophages in the pathogenesis of sepsis.

Staphylococcus aureus is an important human and livestock pathogen. An S. aureus phage (φAvβ) inserted into the chromosome at the beta toxin gene (beta-converting phage) is present in approx. 90 % of human strains and contributes to human-specific innate immune evasion. Comparative genomic analysis of S. aureus isolates from infected poultry has revealed an avian-specific subfamily of beta-converting phages represented by multiple variants with distinct integrase gene alleles. To investigate the role of the avian beta-converting phages in host-pathogen interactions, a φAvβ-deficient strain with non-functional beta-toxinand a φAvβ-deficient strain with restored beta-toxin were constructed by allele replacement in an avian pathogenic S. aureus strain. Compared to the wild type, both φAvβ-deficient strains have reduced net extracellular growth in vitro in chicken bone-marrow derived macrophages. Further investigation using GFP-tagged bacteria has revealed that both φAvβ-deficient strains show reduced initial phagocytosis and intracellular survival compared to the wild type. Absence of φAvβ is also associated with decreased killing of the chicken bone-marrow derived macrophages. We are currently investigating the mechanism underlying this phenotype using deletion mutants of the candidate phage effector genes. Ongoing work also involves using RNAseq to investigate differential host transcriptional response of the macrophages to S. aureus in presence/absence ofφAvβ. Overall, these data will contribute new information relating to the evolution of avian S. aureus and mechanisms of bacterial host-adaptation.

Clostridium difficile is a Gram-positive, spore forming bacterium, which remains a formidable pathogen as the etiological agent of C. difficile infection (CDI). Substantial effort goes into diagnosis of CDI and characterisation of circulating toxigenic C. difficile strains for epidemiology and infection prevention and control. Currently, molecular typing of C. difficile requires 9 days following diagnosis through PCR ribotyping and multilocus variable number tandem repeat (VNTR) analysis. There is a need for more rapid typing methods to investigate possible linkage between CDI cases in healthcare settings. This study developed a one-step, closed tube real-time PCR and high resolution melt (HRM) assay targeting the intergenic spacer region (ISR) and several VNTR loci, with results generated in 2.5 h. The discriminatory power of the PCR-HRM assay was investigated by typing previously characterised toxigenic clinical and animal C. difficile isolates (n=90). Through comparison of HRM profiles targeting the ISR of isolates belonging to 17 PCR ribotypes, 13 HRM genotypes were recognised with 11 PCR ribotypes resolved from each other. Using correlation between HRM data and known VNTR repeat numbers at the B7, C6 and G8 loci, VNTR repeat numbers for isolates could be predicted within an average absolute difference of 1.8 at the B7 locus, 2.1 at the C6 locus, and 2.5 at the G8 locus. These results suggest that a PCR-HRM assay with a multilocus panel targeting ISR and selected VNTR loci could form part of an improved molecular typing scheme for toxigenic C. difficile strains that is faster than currently available methods.

Persister cells are characterised as being viable but non-culturable, a state that preserves their metabolic energy to survive the environmental stress, which allows for recurrent infections. Detection of persisters is, therefore, not possible with standard culture-dependent methods. Furthermore, the effect of antibiotics on the induction of persisters has not been assessed. This study aimed to identify antibiotic-induced persistence and determine the percentage of heterogeneity. Vancomycin, daptomycin and dalbavancin were assessed by standard MIC methods against selected Staphylococcus aureus strains. Replicates of MIC assays were stained with propidium iodide to quantify live/dead and a reactive oxygen species (ROS) dye to detect and quantify persisters using culture-independent single-cell sorting, independently. A comparative analysis was then performed. Dalbavancin showed the lowest MIC values against tested S. aureus strains followed by daptomycin and vancomycin. Cell sorting of vancomycin-, daptomycin- and dalbavancin-treated S. aureus strains showed a range of 1.9–10.2 %, 17.7–62.9 % and 7.5–77.6 % live cells based on the strain, respectively, in which daptomycin, in particular, was a strong inducer of a persister population. Persisters represented 3.7–16 % of the bacterial population. The culture-independent identification of antibiotic-induced persistence through studying at the single-cell level showed different efficacy of antibiotics than standard MIC. Vancomycin was the most effective antibiotic against tested strains followed by dalbavancin then daptomycin as assessed by cell sorting. Therefore, re-evaluation of standard MIC methods may be required to assess the efficacy of antibiotics. Additionally, the detection of daptomycin-associated persisters may provide an elucidation to the reported rapid resistance development in vivo.

The search for alternative, potent, cost-effective treatment of ailments caused by resistant microorganisms and the role of plants and their products as essential sources of medicinal agents is receiving increasing attention. Several of these natural products are reported to have capacity to produce natural compounds of high structural diversity that serve as defense agents against invading microorganisms. Cnidoscolus aconitifolius also known as tree spinach is an indigenous tropical tree that has gained lot of importance in its nutritive value and traditional use. This study evaluates the possible effect of Cnidoscolus aconitifolius leaf extract alone, extract/honey combination on Staphylococcus epidermidis clinical isolate. Phytochemical analysis and antimicrobial efficacy of the extract were performed using standard methods. The results of phytochemical analysis reveal the presence of carbohydrate, tannins, alkaloids, steroids, flavonoids, anthraquinone, saponins and carotenoids. Antimicrobial activities showed inhibition zone values of 8.0±0.1 mm for aqueous extract alone, 9.0±0.1 mm for aqueous extract/honey combination. The finding suggests that C. aconitifolius might be a good source of compounds that can be used to inhibit the growth of Staphylococcus epidermidis pathogen and further supports its popular and wide traditional applications in the treatment of various illnesses. Hence the need for further research to exploit the full potential of C. aconitifolius tree in order to influence their extensive consumption, storage, improvement and production.