Environmental Spores Research Articles

Clostridium difficile ribotypes 027 and 106: clinical outcomes and risk factors

The present study investigates risk factors for onset of Clostridium difficile-associated diarrhoea, specific ribotype and environmental spore contamination in a District General Hospital in South East England. C. difficile isolates were ribotyped from 97 diarrhoeal cases, following detection of C. difficile toxin from faecal specimens by enzyme immunoassay (Health Protection Agency, Southampton). The isolates were tested for various antimicrobial susceptibilities by E-test. Cases were assessed for prior antibiotic use and followed up for clinical outcomes. Controls were matched for age, sex, ward, length of stay and comorbidity to identify any antibiotic risk factors using conditional logistic regression analysis. Environmental sampling on wards was performed with cycloserine-cefoxitin-egg yolk agar. Forty-five percent C.difficile isolates ribotyped as 027, 39% as 106 and 10% as 001. All ribotypes were resistant to ciprofloxacin, erythromycin and cefotaxime but remained susceptible to metronidazole and vancomycin. The crude (death within 28 days) and early (death within 72h) mortalities were 23% and 11% for the 027 strain, whereas for the 106 ribotype they were 11% and 3%, respectively. The case-control study identified ciprofloxacin usage for >7 days as a significant risk factor (adjusted odds ratios of 3.72; 95% CI: 1.38-10.02; P=0.019). Environmental sampling revealed the presence of spores on faecally contaminated equipment such as commodes and bedpan shells, which persisted after cleaning. Ciprofloxacin appears to encourage C.difficile-associated diarrhoea and should be restricted to short courses. Cleaning agents for clinical equipment must have sporicidal activity to prevent cross-transmission.

Invasive Mykosen und Trauma

Perforating injury allows the dispersion of environmental fungi and fungal spores together with miniscule foreign bodies into traumatized tissue where they can multiply and cause invasive infection. Acute invasive fungal infection after open fractures is uncommon. The severity of the fungal infections depends on the type of injury (perforating, presence of foreign material), the body area and the general condition of the patient. Fungal infections of the immuncompetent host are generally localized within the dermis, invasion of the fascia, muscles and the bone is rare. Injury-related fungal infections of the immuncompromised host may lead to rapid invasion and generalization of the fungal infection. The following review will focus on the fungal infections after perforating injuries and open fractures including the invasive mycoses as a direct consequence of the trauma, post-traumatic fungal infections, tropical fungal infections (mycetoma), invasive fungal infections after near-drowning and nosocomial invasive fungal infections of the critically ill post-traumatic patient admitted to the intensive care unit.

Ten-year air sample analysis of Aspergillus prevalence in a university hospital

Airborne fungal samples were collected on a monthly basis for 10 years, from 1995 to 2005, at a tertiary university hospital. Paired samples were cultured at 25 and 37 degrees C. Data were interpreted according to the air filtration systems serving each location. Samples cultured at 37 degrees C from the patient care areas had a mean recovery of 18% of the mean recovery from outdoor air (22 versus 122cfu/m(3)). Recovery of Aspergillus spp. at 37 degrees C in the high-efficiency particulate air (HEPA)-filtered locations was positive for Aspergillus spp. approximately one-third of the time; the rest of the patient care areas were positive half of the time and the outdoor samples were positive 95% of the time. We found 48 sporadic bursts at 37 degrees C which produced counts >3 SD above the mean. Hospital-acquired infection was related to high recovery of Aspergillus fumigatus on at least one occasion. We have found it impossible, without implementing impractical measures, to provide an environment completely devoid of Aspergillus spp. We conclude that routine air sampling is not an effective means of predicting hospital-acquired infections. However, a transient spike, or burst, may be useful in identifying an in-house source of contamination and may be used to consider additional interventional treatments for patients at risk. Emphasis should be placed on maintaining high-efficiency filtration of the outside air and on ensuring that other environmental control methods are used to prevent dissemination of environmental opportunistic fungal spores.

High Resolution FESEM and TEM Reveal Bacterial Spore Attachment

Transmission electron microscopy (TEM) studies in the 1960s and early 1970s using conventional thin section and freeze fracture methodologies revealed ultrastructural bacterial spore appendages. However, the limited technology at that time necessitated the time-consuming process of imaging serial sections and reconstructing each structure. Consequently, the distribution and function of these appendages and their possible role in colonization or pathogenesis remained unknown. By combining high resolution field emission electron microscopy with TEM images of identical bacterial spore preparations, we have been able to obtain images of intact and sectioned Bacillus and Clostridial spores to clearly visualize the appearance, distribution, resistance (to trypsin, chloramphenicol, and heat), and participation of these structures to facilitate attachment of the spores to glass, agar, and human cell substrates. Current user-friendly commercial field emission scanning electron microscopes (FESEMs), permit high resolution imaging, with high brightness guns at lower accelerating voltages for beam sensitive intact biological samples, providing surface images at TEM magnifications for making direct comparisons. For the first time, attachment structures used by pathogenic, environmental, and thermophile bacterial spores could be readily visualized on intact spores to reveal how specific appendages and outer spore coats participated in spore attachment, colonization, and invasion.

Larssoniella duplicati n.sp. (Microsporidia, Unikaryonidae), a newly described pathogen infecting the double-spined spruce bark beetle, Ips duplicatus (Coleoptera, Scolytidae) in the Czech Republic

Larssoniella duplicati n.sp. infects the midgut muscularis, the Malpighian tubules, and the ovaries of adult Ips duplicatus (Sahlb.) in the Czech Republic. The microsporidian attacks up to 50% of the population. Oval spores of two sizes, 3–3.5×1.5–2 and 2–2.5×1.5 μm have the polar filament coiled in 6/7 coils, representing primary and environmental spores, respectively. In early sporogony the young spores produce long electron dense threads and tubules of secretions, which remain fixed around the spore and avoid their free release during dissection of infected hosts. The microsporidian was not found in associated bark beetles such as Ips typographus (L.), or I. amitinus (Eichh.) and others.

Nosema chrysorrhoeae n. sp. (Microsporidia), isolated from browntail moth ( Euproctis chrysorrhoea L.) (Lepidoptera, Lymantriidae) in Bulgaria: Characterization and phylogenetic relationships

A new microsporidian parasite Nosema chrysorrhoeae n. sp., isolated in Bulgaria from the browntail moth ( Euproctis chrysorrhoea L.), is described. Its life cycle includes two sequential developmental cycles that are similar to the general developmental cycles of the Nosema-like microsporidia and are indistinguishable from those of two Nosema spp. from Lymantria dispar. The primary cycle takes place in the midgut tissues and produces binucleate primary spores. The secondary developmental cycle takes place exclusively in the silk glands and produces binucleate environmental spores. N. chrysorrhoeae is specific to the browntail moth. Phylogenetic analysis based on the ssu rRNA gene sequence places N. chrysorrhoeae in the Nosema/ Vairimorpha clade, with the microsporidia from lymantriid and hymenopteran hosts. Partial sequences of the lsu rRNA gene and ITS of related species Nosema kovacevici (Purrini K., Weiser J., 1975. Natürliche Feinde des Goldafters, Euproctis chrysorrhoea L., im Gebiet von Kosovo, FSR Jugoslawien. Anzeiger fuer Schädlingskunde, Pflanzen-Umweltschutz, 48, 11–12), Nosema serbica Weiser, 1963 and Nosema sp. from Lymantria monacha was obtained and compared with N. chrysorrhoeae. The molecular data indicate the necessity of future taxonomic reevaluation of the genera Nosema and Vairimorpha.

Increased resistance of environmental anaerobic spores to inactivation by UV

Water Company Europoort started a pilot plant (MP)UV study to determine the UV-fluence to meet the Dutch drinking water standards. The results of large volume sampling of this pilot plant demonstrated that environmental spores of sulphite-reducing clostridia (SSRC) were highly resistant against UV. With the pilot plant at a flow of 180 m3/h a challenge test was conducted to compare the susceptibility of environmental SSRC and lab-cultured spores of C. perfringens, MS2 bacteriophages and Bacillus subtilis. The latter was dosed as a biodosimeter with a calibrated UV253.7nm sensitivity to confirm the germicidal fluence. This test demonstrated that environmental SSRC were a factor of 1.6 more resistant against UV than the lab-cultured spores of C. perfringens. Furthermore, the results of these environmental SSRC indicated that they are more resistant to UV than other relevant micro-organisms for drinking water safety. Environmental SSRC calibrated with biodosimetry and determined with large volumes sampling is a potential parameter for on-site verification of the efficacy of UV-systems. Further research is necessary to determine to what extent the UV susceptibility of SSRC is consistent in the water and to find the cause of the increased resistance of environmental micro-organisms.

Species-specific peptide ligands for the detection of Bacillus anthracis spores.

Currently available detectors for spores of Bacillus anthracis, the causative agent of anthrax, are inadequate for frontline use and general monitoring. There is a critical need for simple, rugged, and inexpensive detectors capable of accurate and direct identification of B. anthracis spores. Necessary components in such detectors are stable ligands that bind tightly and specifically to target spores. By screening a phage display peptide library, we identified a family of peptides, with the consensus sequence TYPXPXR, that bind selectively to B. anthracis spores. We extended this work by identifying a peptide variant, ATYPLPIR, with enhanced ability to bind to B. anthracis spores and an additional peptide, SLLPGLP, that preferentially binds to spores of species phylogenetically similar to, but distinct from, B. anthracis. These two peptides were used in tandem in simple assays to rapidly and unambiguously identify B. anthracis spores. We envision that these peptides can be used as sensors in economical and portable B. anthracis spore detectors that are essentially free of false-positive signals due to other environmental Bacillus spores.

Unikaryon polygraphi sp.n. (Protista, Microspora): a new pathogen of the four‐eyed spruce bark beetle Polygraphus poligraphus (Col., Scolytidae)

The microsporidium Unikaryon polygraphi sp.n., a pathogen of Polygraphus poligraphus in Austria is described based on light microscopic and ultrastructural characteristics. All life stages have isolated nuclei. Sporogony ends with uninucleate single sporoblasts and spores. Mature oval spores measure 2.5–3.0 μm × 1.0–1.5 μm. The larger spores (3 × 1.5 μm) belong to the `early spore type' with a polar filament coiled in five turns and the smaller spores (2.5 × 1 μm) with polar filament coiled in 6/7 turns belong to the `environmental spore type'. Columnar cells of the midgut, longitudinal and circular muscles and the secretory part of Malpighian tubules of adult beetles are infected. Mature spores are excreted together with the faeces.

Molecular characterization of Nosema bombi (Microsporidia: Nosematidae) and a note on its sites of infection in Bombus terrestris (Hymenoptera: Apoidea)

SUMMARYInvestigations of queen, worker and male bumble bees (Bombus terrestris) showed that all individuals became infected with Nosema bombi. Infections were found in Malpighian tubules, thorax muscles, fat body tissue and nerve tissue, including the brain. Ultrastructural studies revealed thin walled emptied spores in host cell cytoplasm interpreted as autoinfective spores, besides normal spores (environmental spores) intended for parasite transmission between hosts. The nucleotide sequence of the gene coding for the small subunit rRNA (SSU-rRNA) from Microsporidia isolated from B. terrestris, B. lucorum, and B. hortorum were identical, providing evidence that N. bombi infects multiple hosts. The sequence presented here (GenBank Accession no AY008373) is different from an earlier submission to GenBank (Accession no U26158) of a partial sequence of the same gene based on material collected from B. terrestris. It still remains to be investigated if there is species diversity among Microsporidia found in bumble bees.

Evaluating bacterial aerobic spores as a surrogate for Giardia and Cryptosporidium inactivation by ozone

The inactivation kinetics of two strains of bacterial aerobic spores were investigated in a batch reactor for two pH levels (6.3 and 8.2) and various ozone dosages (0.5–2.5 mg/l). The kinetics of ozone decomposition was found to satisfactorily fit a first-order decay model. The CT (concentration×contact time) values were calculated using the modified Hom equation with exponential disinfectant decay. Lower pH resulted in lower CT values. The environmental spores tested were not as resistant to ozone as the reference strain of B. subtilis. Comparison of CT values for aerobic spores with published data suggests that bacterial spores could be a surrogate for evaluating the ozone inactivation of G. lamblia cysts and C. parvum (oo)cysts during drinking water disinfection. However, more information on the resistance of naturally occurring populations of bacterial spores would be needed before they are used as an indicator of ozone disinfection efficiency.

Nosema portugal,N. SP., Isolated from Gypsy Moths (Lymantria disparL.) Collected in Portugal

A microsporidiumNosema portugaln. sp. was isolated from gypsy moths,Lymantria disparL, collected near Lisbon, Portugal, in 1985. The life cycle includes two sequential developmental cycles, a primary and a secondary cycle. The primary cycle occurs in midgut epithelial cells, where primary spores are produced within 48 h. The primary spores immediately extrude their polar filaments, presumably to infect other cells. In the target tissues (salivary glands and fat body) the secondary development cycle is followed by the formation of environmental spores. Primary spores were also sometimes present in target tissues. Fresh unfixed and unstained primary spores have a large posterior vacuole and measured 4.8 × 2.7 μm. Ultrastructurally, they have 5–8 polar filament coils, a large posterior vacuole, abundant endoplasmic reticulum, and were binucleate. Mature unfixed and unstained environmental spores were highly refractive and the posterior vacuole and nuclei could not be seen through the spore coat. Fresh environmental spores measured 4.5 × 1.9 μm. Ultrastructurally, environmental spores were binucleate, with a typical polaroplast, 10–11 isofilar polar filament coils, and a series of 4–6 thin polar filament-like tubules situated at the posterior end of the row of typical polar filament coils. The ssu rRNA sequences strongly suggest that this species is more closely related to theVairimorphasubgroup within theNosema/Vairimorphaclade than to theNosemasubgroup.

Value of environmental sampling and molecular typing of aspergilli to assess nosocomial sources of aspergillosis

In order to investigate the risk of hospital-acquired infections due to environmental Aspergillus, air sampling outside and inside the bone marrow transplantation (BMT) clinic of the University Hospital, Essen, Germany, was performed prospectively for one year. The spore concentration in the air was matched with meteorological data. Two BMT-patients, who were hospitalized during the sampling period, suffered from aspergillosis after discharge. The patients' isolates obtained at re-admission were compared with environmental isolates obtained during the first hospitalization. Analysis by randomly amplified polymorphic DNA showed that the two BMT-patients were infected with Aspergillus strains that were different from the environmental strains. It is concluded that it is not possible to predict the environmental Aspergillus spore concentration by analysis of meteorological data. Since the concentration of specific strains may fluctuate rapidly, a hospital-acquired Aspergillus infection cannot be excluded even if the infecting strain is not found in the hospital environment.

151 Immunoreactive alternaria allergen in house dust: Presence in prairie homes in the absence of environmental mold spores

151 Immunoreactive alternaria allergen in house dust: Presence in prairie homes in the absence of environmental mold spores

Role of Altemaria and Penicillium spores in the pathogenesis of asthma

The ability to harvest spore-rich isolates of molds permitted quantitative studies of their role in the pathogenesis of asthma. Alternaria and Penicillium were selected as examples of ubiquitous molds that readily induce IgE antibodies and are of contrasting sizes. Extracts from those spores were prepared for skin tests and aerosol bronchial challenges. Intact spores were used in the same subjects in bronchial challenges delivered by a Spinhaler. Seven patients with a history of mild asthma received a total of 16 bronchial challenges with the mold to which they had been sensitized. Provocative doses in spore equivalents for a 35% drop in SG aw, 20% drop in FEV 1, or 25% drop in PEER were sought for each challenge. Density dependence-flow rates were also determined. Environmental spore survey data were obtained and compared with the challenge doses for these spores. It was found that (1) immediate-type asthma was readily provoked by both whole spores and by their extracts, (2) in some subjects fewer intact than extracted spores were required, (3) delayed-type asthma occurred only after whole spore challenges, (4) SG aw was the most sensitive and equally specific of the pulmonary function tests, and (5) provocative doses of spore equivalents were within natural exposure ranges. The study confirmed that Alternaria and Penicillium spores in relatively natural states and numbers were potent immunopathogens for asthma.

A guide to the recent literature on aspergillosis as caused by Aspergillus fumigatus, a fungus frequently found in self-heating organic matter

Spores of Aspergillus fumigatus have been found to be abundantly present in the outdoor air at a site where large scale experimental composting of sewage sludge is in progress at Beltsville, Maryland. The health significance of this finding, for that site and for others in the future, is still only incompletely understood. Further studies are in progress to characterize absolute concentrations of the spores of the fungus in air at the site, spore dispersal by air from composting operations, and background environmental spore levels in air. The present paper contains a list of references to papers on health effects of A. fumigatus, many published in the past ten years, along with a review of the same designed to assist the reader in finding information on particular aspects of the subject in the literature. It is intended primarily as an aid to individuals interested in sludge composting and wishing to attain an insight into the A. fumigatus-composting situation, but it may also interest others concerned with other substrates which become moldy at 40--50 C. A. fumigatus has been found in great numbers in naturally and artificially heated environments such as decaying leaves, compost heaps, solar heated sloughs, cooling canals for nuclear power generators, silos, grain storage bins, boiler rooms, detritus around steam turbines and sauna baths. The evident practical merits of sludge composting have been described elsewhere; the information presented here has its main significance in respect to requirements for choice of locations for composting sites and to process and design criteria.