Discovery of inhibition of Burkholderia cenocepacia, Pseudomonas aeruginosa and Stenotrophomonas maltophilia by the Brown Rot Basidiomycete Fungus, Postia placenta.

Antimicrobial resistance (AMR) has now emerged as a major global public health problem. Certain bacterial pathogens, particularly Gram negative organisms associated with patients with cystic fibrosis (CF), have become resistant to several classes of antibiotics resulting in pan-resistance, which creates a clinical treatment dilemma. This study wished to explore the production of antibacterial extracellular metabolites from plant pathogenic fungi. Fungal Culture Extracts (FCEs) were prepared from 10 fungi (Armillaria gallica, Clitocybe nebularis, Fusarium coeruleum, Fusarium oxysporum, Fusarium poae, Hymenoscyphus fraxineus, Nectria fuckeliana, Phytophthora infestans, Phytophthora ramorum, Postia placenta), which were tested for activity against the CF pathogens, Pseudomonas aeruginosa (PA) (n=8), Burkholderia cenocepacia (n=2) and Stenotrophomonas maltophilia (n=2). In addition, FCE were assessed for their ability to alter antibiotic susceptibility in PA (n=8), with six antipseudomonal antibiotics (ceftazidime, ciprofloxacin, colistin, meropenem, piperacillin/tazobactam, tobramycin). None of the FCEs showed inhibitory activity to the 12 bacterial isolates tested, with the exception of the FCE from Postia placenta, which showed inhibition against all 12 bacteria. An antagonistic interaction was observed, where a statistically significant decrease in mean zone sizes was noted with Armillaria gallica (p=0.03) and Phytophthora infestans (p=0.03) FCEs and their interaction with the fluoroquinolone antibiotic, ciprofloxacin. Given the increase in clinical morbidity and mortality associated with chronic lung infections with Pseudomonas aeruginosa, Burkholderia cenocepacia and Stenotrophomonas maltophilia, coupled with the difficulty in treating such chronic infection due to overwhelming antimicrobial resistance, any novel substance showing inhibition of these organisms merits further investigation as a potential future antimicrobial agent, with potential clinical therapeutic application.

Antimicrobial properties of phytohormone (gibberellins) against phytopathogens and clinical pathogens.

The in vitro antimicrobial potential of physiologically active diterpenoid plant-derived gibberellins (gibberellic acids; GAs) was tested on microbial pathogens of significance to plant and human health. The racemic enantiomer GA3 produced varying inhibitory effects against a wide range of plant host disease causal agents (phytopathogens) comprising fungi, oomycetes and bacteria. The results showed that GA3 effected either strong growth arrest of phytopathogenic fungi or holistic biocidal effects on oomycete and phytopathogenic fungi at higher concentration (>10-50 mM) and increased hyphal extension growth when the concentration of GA3 was lowered (<10-0.1 mM). When human clinical pathogenic bacteria cohorts were challenged with gibberellin enantiomers, viz GA1, GA4, GA5, GA7, GA9 and GA13 (50 mM), employing Kirby-Bauer disc bioassay methods for assessment of their efficacies, no inhibitory effect was seen with gibberellin enantiomers, viz GA1, GA3, GA5 and GA13, while GA4 inhibited all human clinical bacterial organisms examined, with GA7 and GA9 showing limited activity. The antibiotic effects of enantiomeric diterpenoid phytohormones evinced in our preliminary study raise prospects for further studies to fully examine their potential therapeutic value for human healthcare and their compliance with cytotoxicity and other ethical considerations in the future.

Antimicrobial properties of basidiomycota macrofungi to Mycobacterium abscessus isolated from patients with cystic fibrosis.

BACKGROUND: Antimicrobial resistance (AMR) has now emerged as a global public health crisis. Of particular concern is AMR associated with the genus Mycobacterium, including Mycobacterium tuberculosis and the nontuberculous mycobacteria (NTM). Emergence of the NTM, in particular Mycobacterium abscessus, in patients with cystic fibrosis (CF) represents both a diagnostic and a treatment dilemma. Such resistance drives the need to investigate novel sources of antimicrobials. Medicinal fungi have a well-documented history of use in traditional oriental therapies. Not only is this an ancient practice, but also still today, medical practice in Japan, China, Korea, and other Asian countries continue to rely on fungal-derived antibiotics. A study was, therefore, undertaken to examine the antimicrobial activity of 23 native macrofungal (mushrooms/toadstools) taxa, collected from woodlands in Northern Ireland against six clinical (CF) isolates of M. abscessus, as well as M. abscessus National Collection of Type Cultures (NCTC) Reference strain (NCTC 13031). METHODS: Free-growing saprophytic and mycorrhizal macrofungi (n = 23) belonging to the phylum Basidiomycota were collected and were definitively identified employing Polymerase Chain reaction/ITS DNA sequencing. Macrofungal tissues were freeze-dried and reconstituted before employment in antibiotic susceptibility studies. RESULTS: All macrofungi examined showed varying inhibition of the M. abscessus isolates examined with the exception Russula nigricans. The macrofungi displaying maximum antimycobacterial activity against the clinical isolates were (in descending order) M. giganteus (33.6 mg/ml), Hygrocybe nigrescens (38.5 mg/ml) and Hypholoma fasciculare (25.3 mg/ml). CONCLUSION: Macrofungi may represent a source of novel antimicrobials against M. abscessus, which have not yet been fully explored nor exploited clinically. This is the first report describing the antimycobacterial properties of extracts of M. giganteus against M. abscessus. Further work is now required to identify the constituents and mode of the inhibitory action of these macrofungi against the M. abscessus. Given the gravity of AMR in the NTMs, particularly M. abscessus and the clinical treatment dilemmas that such AMR present, antibiotic drug discovery efforts should now focus on investigating and developing antibacterial compounds from macrofungi, particularly M. giganteus, where there are no or limited current treatment options.

Antimicrobial activity of Calendula officinalis petal extracts against fungi, as well as Gram-negative and Gram-positive clinical pathogens.

The aim of the present study was to assess the antimicrobial activity of methanol and ethanol extracts of pot marigold (Calendula officinalis) petals against clinical pathogens. The antimicrobial potential of C. officinalis extracts was evaluated against a panel of microorganisms isolated from patients at the Belfast City Hospital (BCH), including bacteria and fungi, using disc diffusion assay. Methanol extract of C. officinalis exhibited better antibacterial activity against most of the bacteria tested, than ethanol extract. Both methanol and ethanol extracts showed excellent antifungal activity against tested strains of fungi, while comparing with Fluconazole.

Bacterial stress response to environmental radiation relating to the Fukushima radiation discharge event, Japan: will environmental bacteria alter their antibiotic susceptibility profile?

Antibiotic resistance in clinical pathogens in humans may be traced back to resistance mechanisms in environmental bacteria and any factors, which are likely to alter (upregulate) resistance in environmental organisms, is of potential and eventual consequence to human pathogens. Furthermore, sublethal doses of gamma radiation to environmental organisms may cause sublethal stress and a selective pressure, which may lead to mutational events that alter the bacterium's susceptibility profile. A gamma (γ) radiation simulation experiment was performed to emulate the exposure of four environmental bacteria, including Listeria innocua, Bacillus subtilis, E. coli and Pseudomonas aeruginosa, to levels of radiation in and around Fukushima, Japan, equating to 1, 10 and 100 years equivalence exposure. Alteration to susceptibility to 14 antibiotics was measured as the primary endpoint. There was no significant alteration in the susceptibility of the Gram-positive organisms, whereas both Gram-negative organisms became slightly more susceptible to the antibiotics tested over time. These data indicate that such radiation exposure will not increase the antibiotic resistance profile of these organisms and hence not add to the global public health burden of increased antibiotic resistance in human bacterial pathogens.

Molecular identification of airborne bacteria associated with aerial spraying of bovine slurry waste employing 16S rRNA gene PCR and gene sequencing techniques.

Polymerase chain reaction amplification of the universal 16S ribosomal RNA (rRNA) gene was performed on a collection of 38 bacterial isolates, originating from air sampled immediately adjacent to the agricultural spreading of bovine slurry. A total of 16 bacterial genera were identified including both Gram-positive and Gram-negative genera. Gram-positive organisms accounted for 34/38 (89.5%) of total bacterial numbers consisting of 12 genera and included Staphylococcus (most common genus isolated), Arthrobacter (2nd most common genus isolated), Brachybacterium, Exiguobacterium, Lactococcus, Microbacterium and Sporosarcina (next most common genera isolated) and finally, Bacillus, Brevibacterium, Frigoribacterium, Mycoplana and Pseudoclavibacter. Gram-negative organisms accounted for only 4/38 (10.5%) bacterial isolates and included the following genera, Brevundimonas, Lysobacter, Psychrobacter and Rhizobium. No gastrointestinal pathogens were detected. Although this study demonstrated a high diversity of the microorganisms present, only a few have been shown to be opportunistically pathogenic to humans and none of these organisms described have been described previously as having an inhalational route of infection and therefore we do not believe that the species of organisms identified pose a significant health and safety threat for immunocompetant individuals.

An examination of antibacterial and antifungal properties of constituents described in traditional Ulster cures and remedies.

Traditional herbal cures and remedies have played an important historical role in the treatment of a variety of illnesses and diseases in Northern Ireland for the last three hundred years. Recently, these have been reviewed in the publication by Linda Ballard from the Ulster Folk and Transport Museum at Cultra, Co. Down, which details the variety of local plants used and for what purpose. From this publication and another related publication, we note the description of several plant species that consistently appear in traditional cures and remedies, particularly used to treat infections and infectious diseases. Unfortunately, although these plants have strong associations with the local historical evidence base, there are very limited and mainly no formal publications in the medical/scientific evidence base, examining their scientific background and clinical efficacy.

Avian influenza, migratory birds and emerging zoonoses: Unusual viral RNA, enteropathogens and Cryptosporidium in poultry litter.

The last decade has witnessed the emergence of several new viral infectious agents, most notably avian influenza H5N1, SARS and West Nile Virus. The emergence of these agents is heavily associated with zonotic animal hosts, as well as migratory pathways of infected bird vectors. The environmental survival and persistence of nucleic acid associated with these viral agents may be important for both the detection as well as the occurrence of related diseases. Our hypothesis suggests that nucleic acid from such emerging viruses may enter into a virus-parasite surrogate relationship to aid in viral persistence. We suggest that Cryptosporidium and other gastrointestinal parasites, including Giardia, could be a) a reservoir of genetic material and a environment where assortment between that genetic variation can occur and, b) a source of zoonoses through infection of the 'target' animal (including humans). One example which illustrates this may be the uptake dsRNA from rotavirus into cryptosporidial oocysts, as this parasite has previously been shown to contain dsRNA viral-like particles. The importance of such a surrogate relationship is discussed and its implications for human and animal health highlighted.

A novel challenge test incorporating irradiation (60Co) of compost sub-samples to validate thermal lethality towards pathogenic bacteria.

Maturing compost heaps normally attaining temperatures ranging from 55 to 65 degrees C is generally regarded to conform to recommended biological risks and sanitation standards for composts stipulated by either EU or US-EPA. Composted products derived from animal sources are further required by EU biohazard safety regulatory legislation that such composts either attain 70 degrees C for over 3h during maturation or via treatment at 70 degrees C for 1h before being considered for dispensation on land. The setting of the upper limit of thermal lethality at 70 degrees C/1h for achieving biosecurity of the animal waste composted products (e.g. pelleted fertilizer formulations) is not properly substantiated by specific validation tests, comprising a 'wipe-out' step (usually via autoclaving) followed by inoculation of a prescribed bacterium, exposure to 70 degrees C/1h and the lethality determined. Pelleted formulations of composts are not amenable for wet methods (autoclaving) for wipe-out sterilization step as this is detrimental to the pellet and compromises sample integrity. This study describes a laboratory method involving the employment of ((60)Co) irradiation 'wipe-out' step to: (a) compost sub-samples drawn from compost formulation heaps and (b) pelleted products derived from composted animal products while determining the thermal lethality of a given time/temperature (70 degrees C/1h) treatment process and by challenging the irradiated sample (not just with one bacterium but), out with 10 potential food-poisoning organisms from the bacterial genera (Campylobacter, Escherichia, Listeria, Salmonella, Yersinia) frequently detected in pig and poultry farm wastes. This challenge test on compost sub-samples can be a useful intervention ploy for 'inspection and validation' technique for composters during the compost maturity process, whose attainment of temperatures of 55-65 degrees C is presumed sufficient for attainment of sanitation. Stringent measures are further required by law for composted products arising from rural industrialists producing pelleted fertilizers from re-composted animal agriculture wastes comprising pig slurry solids, poultry litter and spent mushroom compost, which carry residual food-borne pathogens with implications to the food chain including humans. Environmentally, sustainable means of recycling farm wastes require that final composted products are free of pathogens in compliance with environmental safety legislation before their release to the market. This test developed provides a science-based risk characterization tool for sustainably managing environmental safety by 'validating' thermal lethality of a given composting process or their derivatives achieved without compromising the sample integrity or ambiguity attached to microbiological validation involving steam sterilization or autoclaving procedures and helps audit the resurgent bacterial populations from surviving non-pathogenic organisms in the end-products of animal waste compost formulations.

Antibacterial activity of honey against community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA).

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has now been described globally, as a clinically significant pathogen, particularly associated with skin and soft tissue infections, including abscesses, cellulitis and furunculosis. The recent emergence of CA-MRSA combined with its predominant presentation associated with skin and soft tissue infection, the previous literature indicating honey as an effective treatment of healthcare-associated HA-MRSA-related wound infection, as well as honey's ease of topical application, make the current study timely and of interest to healthcare practitioners involved with wound management. Although previous studies have examined the antimicrobial activity of honey against HA-MRSA, such data are limited regarding the activity of honey against this emerging type of MRSA. CA-MRSA (n=6 isolates), was examined for its susceptibility to natural honey (n=3 honey produced from bees in Northern Ireland and one commercial French honey). Results demonstrated that all honey was able to reduce the cultural count of all CA-MRSA from approximately 10(6) colony-forming units (cfus) (mean = 6.46 log10 cfu/g) to none detectable within 24h of co-culture of separate CA-MRSA organisms individually with all four-honey types examined. Subsequent non-selective enrichment of honey demonstrated that inoculated honey remained positive for CA-MRSA until 72h postinoculation, after which point no culturable organisms could be detected. This study demonstrated that, in vitro, these natural products had an antimicrobial activity against the CA-MRSA organisms tested. Further studies are now required to demonstrate if this antimicrobial activity has any clinical application.

The enigma of double-stranded RNA (dsRNA) associated with mushroom virus X (MVX).

New variants of pathogenic fungal viruses are emerging and they are enigmatic in revealing their molecular identity and of their origin. Double-stranded RNAs, some in non-encapsidated forms are increasingly becoming causal agents for sporadic diseases and are consistently associated with a complex profile of dsRNAs, presumably of (multiple) viral origin present in the host while the same are conspicuously absent in healthy (looking) counterparts. The emergence of an unusual Agaricus bisporus mushroom 'patch disease' first reported in 1996, later termed as 'mushroom virus X' (MVX), exhibited a wide range of symptoms (e.g. barren patches beside healthy looking mushrooms, arrested pins, premature opening, brown, off-colour and distortions in shape). A variable compendium of novel 26 (dsRNA) elements, ranging in sizes between 20.2kb to 0.64kb, several of them (-17/26) in non-encapsidated form have been shown to occur in the diseased mushroom fruiting bodies and are thought to comprise multiple viruses. Ten years on, this devastating disease is now more widespread and prevalent in a number of European countries (e.g. The Netherlands, Ireland) ranging from occasional to severe outbreaks leading to crop losses. Impressive data has been accumulated on the symptoms, but the potential aetiological sources, biochemical and molecular characterizations corresponding to the symptoms vis-a-vis MVX linked dsRNAs still remain either elusive or unclear. We have overviewed mainly the molecular findings of research groups working on MVX in these countries together with our own work on MVX in Northern Ireland. To date, the results reviewed suggest that with the exception of 4 low molecular weight dsRNA bands (sizes 2.0. 1.8, 0.8 and 0.6 kb) which consistently were found synchronous to mushroom off-colour/browning symptoms in the UK and The Netherlands, other individual MVX dsRNAs or their banding patterns clearly lack credible relationship with other symptoms of the MVX disease complex. The issues in the molecular characterisation of the MVX dsRNAs include the disparate results on the molecular sequences obtained for some of these by the different research groups, the varying molecular methods or approaches adopted by them for deciphering the nucleotide sequences of the novel dsRNAs that are different from previously encountered mushroom viruses. The future outlook and general consensus among mushroom researchers worldwide is for an urgent need to recruit international taskforce and re-focus on clarifying the symptom vis-a-vis dsRNAs in the enigmatic MVX disease complex. As crossing the cellular membrane is a key step to infection process, we have also attempted to draw parallels with other viruses in terms of the potential cell entry mechanisms for MVX dsRNAs. In the light of MVX disease and A. bisporus being a commercial crop worldwide in agri-food markets, and taking cue from its nearest Basidiomycete model mushroom, Coprinus cinereus whose genome mapping is completed, we also propose that it may be timely for the international research groups to renew efforts to prop up a network for sequencing the host A. bisporus mushroom genome (-38 MB) for a better understanding of host-pathogen relationships.

Pelleted organo-mineral fertilisers from composted pig slurry solids, animal wastes and spent mushroom compost for amenity grasslands.

In Ireland, conversion of biodegradable farm wastes such as pig manure spent mushroom compost and poultry litter wastes to pelletised fertilisers is a desirable option for farmers. In this paper, results obtained from the composting of pig waste solids (20% w/w) blended with other locally available biodegradable wastes comprising poultry litter (26% w/w), spent mushroom compost (26% w/w), cocoa husks (18% w/w) and moistened shredded paper (10% w/w) are presented. The resulting 6-mo old 'mature' composts had a nutrient content of 2.3% total N, 1.6% P and 3.1% K, too 'low' for direct use as an agricultural fertiliser. Formulations incorporating dried blood or feather meal amendments enriched the organic N-content, reduced the moisture in mature compost mixtures and aided the granulation process. Inclusion of mineral supplements viz., sulphate of ammonia, rock phosphate and sulphate of potash, yielded slow release fertilisers with nutrient N:P:K ratios of 10:3:6 and 3:5:10 that were suited for amenity grasslands such as golf courses for spring or summer application and autumn dressing, respectively. Rigorous microbiological tests carried out throughout the composting, processing and pelletising phases indicated that the formulated organo-mineral fertilisers were free of vegetative bacterial pathogens.

Cryptosporidiosis in developing countries.

Globally, Cryptosporidium infection continues to be a significant health problem where it is recognized as an important cause of diarrhoea in both immunocompromised and immunocompetent people. In developing countries persistent diarrhoea is the leading cause of death in children younger than five years of age, where it accounts for 30 to 50 percent of those deaths. Encouragingly an increasing number of investigations in developing countries employ molecular tools, significantly improving the quality of epidemiological information. This improved Cryptosporidium monitoring, with appropriate molecular methods, in surface water, livestock, wildlife and humans, will increase current knowledge of infection and transmission patterns, and ultimately help to control Cryptosporidium via improved risk assessments in the future.

Prevalence and identity of Cryptosporidium spp. in pig slurry.

Cryptosporidium spp. were detected in 25 of 56 pig slurry samples from 33 Irish farms by PCR and DNA sequencing. The organisms detected included C. suis, Cryptosporidium pig genotype II, and C. muris. We concluded that Cryptosporidium oocysts can persist in treated slurry and potentially contaminate surface water through improper discharge or uncontrolled runoff.

The epidemiology of antibiotic resistance in Campylobacter.

Antibiotic resistance, particularly with the fluoroquinolones and macrolide antibiotics, has now emerged globally with thermophilic campylobacters, including Campylobacter jejuni and C. coli, giving rise to concerns about how these organisms have acquired such resistance characteristics, as well as consequences for human and animal treatment. This review examines (i) the clinical epidemiology of antibiotic resistance in human and animal thermophilic campylobacters, (ii) an update on resistance rates globally, (iii) surveillance of antimicrobial resistance in campylobacters originating from animals, particularly poultry, (iv) the role of the environment in the acquisition and transmission of antibiotic-resistant campylobacters, as well as (v) issues of biocide resistance in campylobacters.

Campylobacter.

Species within the genus, Campylobacter, have emerged over the last three decades as significant clinical pathogens, particularly of human public health concern, where the majority of acute bacterial enteritis in the Western world is due to these organisms. Of particular concern are the species, C. jejuni and C. coli, which are responsible for most of these gastrointestinal-related infections. Although these organisms have already emerged as causative agents of zoonoses, several aspects of their epidemiology and pathophysiology are only beginning to emerge. Trends in increasing antibiotic resistance are beginning to emerge with oral antibiotics, which may be the drug of choice for when it is necessary to intervene chemotherapeutically. This review wishes to examine (i) emerging clinical aspects of the disease, such as Guillain Barre syndrome (GBS), (ii) the association between these organisms and poultry as a natural host, (iii) environmental aspects of Campylobacter epidemiology, (iv) the emergence of atypical campylobacters (v) emerging trends in antibiotic resistance, (vi) adoption of modern methods for the detection of campylobacters.

Hypersensitivity pneumonitis associated with mushroom worker's lung: an update on the clinical significance of the importation of exotic mushroom varieties.

Hypersensitivity pneumonitis remains an important industrial disease in mushroom workers. It has a significant morbidity, and early diagnosis and removal from exposure to the antigen are critically important in its management. Recently, several new allergens have been described, particularly those from mushroom species originating in the Far East, which are of clinical significance to workers occupationally exposed to such allergens in cultivation, picking, and packing of commercial mushroom crops. Importing of exotic mushrooms including Shiitake is common in EU countries, and some of the exotic species of mushrooms are cultivated for local markets. This practice may contribute to an increase in clinical cases of mushroom hypersensitivity pneumonitis. This update reviews the recent literature and examines changing trends of mushroom worker's lung, with increased movement of commercial product and labour markets worldwide.