MicrobiologyBytes

Candida infections are the fourth most common cause of nosocomial blood stream infections and are associated with a significant mortality. Delays in antifungal therapy have been associated with increased hospital costs of over US$6,000 per patient and overall mortality. The role of HMG CoA reductase inhibitors (statins) in improving outcomes bacteremic sepsis is currently being debated with recent papers showing significantly improved survival in patients with systemic inflammatory response syndrome in the intensive care unit, in patients with chronic kidney renal disease and patients with community acquired pneumonia and influenza. One explanation of this effect is that statins in animal models have shown to reduce inflammatory markers, in particular the release of cytokines and cytotoxic effects of neutrophils. The reduction in inflammatory cytokines has also been demonstrated in patients in a prospective randomized study comparing simvastatin to placebo where there was a significant reduction in tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) in the statin group. Yeasts use the same HMG CoA reductase as humans, however their end-product is ergosterol rather than cholesterol. In vitro studies have demonstrated that simvastatin greatly inhibits the growth of Candida species. This review suggests there is a clinical benefit of statin therapy throughout antifungal therapy in intensive care unit patients with confirmed candidemia.

Statins in Candidemia: clinical outcomes from a matched cohort study. 2010 BMC Infectious Diseases 10: 152 doi:10.1186/1471-2334-10-15210
HMG CoA reductase inhibitors (statins) in patients with bacteremic sepsis have shown significant survival benefits in several studies. There is no data on the effect of statins in candidemic patients, however in-vitro models suggest that statins interfere with ergesterol formation in the wall of yeasts.
This retrospective matched- cohort study from 1/2003 to 12/2006 evaluated the effects of statins on patients with candidemia within intensive care units. Statin-users had candidemia as a cause of their systemic inflammatory response and were on statins throughout their antifungal therapy, while non-statin-users were matched based on age +/- 5 years and co-morbid factors. Primary analysis was 30-day survival or discharge using bivariable comparisons. Multivariable comparisons were completed using conditional logistic regression. All variables with a p-value less than 0.10 in the bivariable comparisons were considered for inclusion in the conditional logistic model.
There were 15 statin-users and 30 non-statin users that met inclusion criteria, all with similar demographics and co-morbid conditions except the statin-users had significantly more coronary artery disease (P<0.01), peripheral vascular disease (P=0.03) and lower median APCAHE II scores (p=0.03). There were no differences in duration of candidemia , antifungal therapy or Candida species between the groups. Statins were associated with lower mortality on bivariable and multivariable analyses compared to controls; although, in the latter the protective effect lacked statistical signficance.
In our small, single-center matched cohort study, statins appear may provide a survival benefit in candidemia, however further studies are warranted to validate and further explore this association.

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Bats, which are ‘keystone species’ in many ecosystems, play notable roles in plant pollination, forest regeneration and control of insect populations. Bats are important to human health as they are reservoirs or carriers for rabies and other viruses, parasites, and pathogenic fungi. Hibernation is believed to be an important adaptation in bats that may contribute to their exceptional longevity. The common little brown bat (Myotis lucifugus) hibernates, along with the endangered Indiana bat (Myotis sodalis), in many hibernacula in the Northeastern United States, including caves and mines in upstate New York. Hibernating bats can suffer significant mortality due to adverse environmental conditions such as freezing or flooding, as well as human activities including visitation and pesticide applications. No mass mortality was reported until recently from bat sites that had been surveyed for almost three decades by the New York State Department of Environmental Conservation. Recently, however little brown bats have been found to be dying in large numbers at many hibernation sites in upstate New York. This problem has spread to other States in the Northeastern USA.

Morphological and Molecular Characterizations of Psychrophilic Fungus Geomyces destructans from New York Bats with White Nose Syndrome (WNS). PLoS ONE 5(5): e10783. doi:10.1371/journal.pone.0010783
Massive die-offs of little brown bats (Myotis lucifugus) have been occurring since 2006 in hibernation sites around Albany, New York, and this problem has spread to other States in the Northeastern United States. White cottony fungal growth is seen on the snouts of affected animals, a prominent sign of White Nose Syndrome (WNS). A previous report described the involvement of the fungus Geomyces destructans in WNS, but an identical fungus was recently isolated in France from a bat that was evidently healthy. The fungus has been recovered sparsely despite plentiful availability of afflicted animals.
We have investigated 100 bat and environmental samples from eight affected sites in 2008. Our findings provide strong evidence for an etiologic role of G. destructans in bat WNS. (i) Direct smears from bat snouts, Periodic Acid Schiff-stained tissue sections from infected tissues, and scanning electron micrographs of bat tissues all showed fungal structures similar to those of G. destructans. (ii) G. destructans DNA was directly amplified from infected bat tissues, (iii) Isolations of G. destructans in cultures from infected bat tissues showed 100% DNA match with the fungus present in positive tissue samples. (iv) RAPD patterns for all G. destructans cultures isolated from two sites were indistinguishable. (v) The fungal isolates showed psychrophilic growth. (vi) We identified in vitro proteolytic activities suggestive of known fungal pathogenic traits in G. destructans.
Further studies are needed to understand whether G. destructans WNS is a symptom or a trigger for bat mass mortality. The availability of well-characterized G. destructans strains should promote an understanding of bat–fungus relationships, and should aid in the screening of biological and chemical control agents.

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Emerging and reemerging infectious diseases are increasing worldwide and represent a major public health concern. One class of emerging human and animal diseases is caused by fungi. A new study examines an outbreak of a fungal infection, Cryptococcus gattii, in the Pacific Northwest of the United States. This fungus has been considered a tropical fungus, but emerged to cause an outbreak in the temperate climes of Vancouver Island in 1999 that is now causing disease in humans and animals in the United States. Because of the way an Oregon-specific strain of the fungus is reproducing and spreading, it will likely move into California and other adjacent areas. This novel fungus is worrisome because it appears to be a threat to otherwise healthy people. Typically, we more often see this fungal disease associated with transplant recipients and HIV-infected patients, but that is not what we are seeing in this case. VGIIc, the new Oregon strain, has yielded dozens of isolates from many specimens, including domesticated animals like cats, dogs and sheep – even an unlucky alpaca. Most of those are nonmigratory animals, which suggests that the animals probably didn’t bring the pathogen from some other region but, rather, acquired it locally. Using molecular techniques, the geneticists uncovered clues that showed the Oregon-only fungal strain most likely arose recently, parallel to the outbreak of C. gattii that began in Canada in 1999 that has now spread into Washington and Oregon.

The researchers found that the novel genotype (VGIIc) is now a major source of C. gattii illness in Oregon. Because C. gattii types had previously been found in tropical areas, the authors speculate that environmental changes may be responsible for the evolution and emergence of this pathogen. Determining the exact origin of the VGIIc type is difficult, and sampling thus far has failed to turn up isolates in Oregon soil, water or trees. The mortality rate for recent C. gattii cases in the Pacific Northwest is running at approximately 25 percent, or 5 out of 21 cases analyzed in the United States, compared to a mortality rate of 8.7 percent of 218 cases in British Columbia, Canada. Most C. gattii infections follow a more complicated clinical course in people than does the more common Cryptococcus neoformans. Symptoms can appear two to several months after exposure, and while most people never develop symptoms, those infected may have a cough lasting weeks, sharp chest pain, shortness of breath, headache (related to meningitis), fever, night time sweats and weight loss. In animals the symptoms are a runny nose, breathing problems, nervous system problems and raised bumps under the skin. While C. gattii can be treated, it cannot be prevented; there is no vaccine. Because the strain is so virulent when it infects some humans and animals, the researchers are calling for greater awareness and vigilance in testing. Some strains of C. gattii are not more virulent than C. neoformans, for example, but doctors need to know what type they are dealing with.

Emergence and Pathogenicity of Highly Virulent Cryptococcus gattii Genotypes in the Northwest United States. PLoS Pathog 6(4): e1000850. doi:10.1371/journal.ppat.1000850
Cryptococcus gattii causes life-threatening disease in otherwise healthy hosts and to a lesser extent in immunocompromised hosts. The highest incidence for this disease is on Vancouver Island, Canada, where an outbreak is expanding into neighboring regions including mainland British Columbia and the United States. This outbreak is caused predominantly by C. gattii molecular type VGII, specifically VGIIa/major. In addition, a novel genotype, VGIIc, has emerged in Oregon and is now a major source of illness in the region. Through molecular epidemiology and population analysis of MLST and VNTR markers, we show that the VGIIc group is clonal and hypothesize it arose recently. The VGIIa/IIc outbreak lineages are sexually fertile and studies support ongoing recombination in the global VGII population. This illustrates two hallmarks of emerging outbreaks: high clonality and the emergence of novel genotypes via recombination. In macrophage and murine infections, the novel VGIIc genotype and VGIIa/major isolates from the United States are highly virulent compared to similar non-outbreak VGIIa/major-related isolates. Combined MLST-VNTR analysis distinguishes clonal expansion of the VGIIa/major outbreak genotype from related but distinguishable less-virulent genotypes isolated from other geographic regions. Our evidence documents emerging hypervirulent genotypes in the United States that may expand further and provides insight into the possible molecular and geographic origins of the outbreak.

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Whether it is in the setting of disease or in a healthy state, the human body contains a diverse range of microorganisms, including bacteria and fungi. The interactions between these taxonomically diverse microorganisms are highly dynamic and dependent on a multitude of microorganism and host factors. Human disease can develop from an imbalance between commensal bacteria and fungi or from invasion of particular host niches by opportunistic bacterial and fungal pathogens. This Review describes the clinical and molecular characteristics of bacterial–fungal interactions that are relevant to human disease.

Medically important bacterial–fungal interactions. 2010 Nature Reviews Microbiology 8, 340-349 doi:10.1038/nrmicro2313

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Actinobacteria and filamentous fungi are just two examples of organisms that use cell elongation for growth. Mathematical models have been used to describe species-specific and more generic polarized growth. In this article in Microbiology Today (pdf) Fordyce Davidson suggests that multi-scale modelling could be the most effective approach to help us understand this morphological phenomenon:

Growth by cell elongation is a morphological process that transcends taxonomic kingdoms. Examples include hyphal tip growth in actinobacteria and filamentous fungi, plant root-hair formation and the development of neurons in animals. Such structures have developed almost certainly because they afford an evolutionary advantage – producing a growth habit well-suited to physically complex environments, facilitating the (internal) redeployment of nutrients or enabling the transfer of information over long spatial scales. The biology involved in producing this polarized growth form is clearly very different in plant, bacterial, fungal or mammalian cells. But its ubiquitous nature suggests that certain ‘”rules” are being followed. Moreover, if we compare fungi and actinobacteria, the foci of this article, it is clear that these rules are scalable: tip growth is similar, irrespective of the orders of magnitude difference in cell size. It appears that these rules form a blueprint for polarized growth.

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Invasive fungal infections are an increasing threat to human health. In the developed world, these infections predominantly occur in increasingly aggressive immunosuppressive therapies. The overall mortality for invasive diseases caused by Candida spp. and Aspergillus spp. is 30–50%, despite the advent of new diagnostic and therapeutic strategies. In the developing world, there are 1 million cases of cryptococcal disease per year, resulting in 675,000 deaths. Allergic fungal syndromes are increasingly recognised. Continued efforts are required to improve the often suboptimal therapeutic outcomes associated with fungal infections.

The high degree of phylogenetic relatedness between fungi and humans means that there are relatively few differential targets to be exploited for antifungal drug development. Fungi are involved in an interminable struggle for survival with each other and with other microbes. They produce a vast array of extracellular enzymes and secondary metabolites to counteract and digest the external world. Many antimicrobial agents have been isolated from fungi themselves. The best example is penicillin, which was isolated from Penicillium notatum (now Penicillium chrysogenum) by Fleming, and later purified for medical use by Florey and Chain. Similarly, the echinocandins, a novel class of antifungal compounds now in widespread clinical use, are semisynthetic derivatives of fungal-derived cyclic hexapeptides. Currently, five classes of antifungal agents are used orally or intravenously for the treatment of fungal infections in humans: polyenes, pyrimidine analogues, allylamines, azoles and the echinocandins. Each antifungal compound has advantages and limitations related to its spectrum of activity, route of administration, drug interactions and toxicity profile.

This review describes the role and limitations of these agents for the treatment of the most common and medically important syndromes, and provides a perspective on the current status of drug therapy for invasive fungal diseases, together with priorities for the future development of novel compounds. Key opportunities for new drugs include production of orally bioavailable agents for the treatment of invasive aspergillosis, invasive candidiasis, cryptococcal meningitis and mucosal and urinary Candida infections. Orally bioavailable agents for the treatment of chronic pulmonary and allergic aspergillosis are also required, as well as new potent drugs against a range of medically important moulds. Antifungal resistance is a problem in certain contexts, but is generally less of a problem than bacterial infections. Earlier and more complete mycological diagnosis and improvements in underlying risk estimation will improve outcomes.

Therapy for fungal diseases: opportunities and priorities. Trends Microbiol. March 5, 2010

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In 1518, one of the strangest epidemics in recorded history struck the city of Strasbourg. Hundreds of people were seized by an irresistible urge to dance, hop and leap into the air. In houses, halls and public spaces, as fear paralyzed the city and the members of the elite despaired, the dancing continued with mindless intensity. Seldom pausing to eat, drink or rest, many of them danced for days or even weeks. And before long, the chronicles agree, dozens were dying from exhaustion. What was it that could have impelled as many as 400 people to dance, in some cases to death?

Medieval dancing epidemics were not unrelated events: they were linked both in time and space. Every one of the ten or so outbreaks between the late 1300s and 1518 happened along the Rhine and Mosel rivers. In 1374, for instance, the crazed dance gradually spread out from an epicentre around Aachen, Liege and Maastricht to neighbouring towns such as Ghent, Utrecht, Metz, Trier and, eventually, Strasbourg. Moreover, outbreaks of compulsive dancing virtually always struck in or close to places affected by earlier outbreaks. Maastricht, Trier, Zurich and Strasbourg each experienced two or more episodes. There are also several reports of compulsive dancing after 1518. All of these, crucially, took place close to the Rhine, and all but one within a short ride of Strasbourg itself.

How can we explain this striking epidemiological picture? One suggestion is that wild dancing formed part of the ecstatic ritual of a heretical sect, an energetic counterpart of the flagellant’s cult. There are two main difficulties with this theory. First, in lucid moments the dancers implored bystanders and priests to come to their aid. There is absolutely no evidence that the dancers wanted to dance. On the contrary, they expressed fear and desperation. Second, the authorities consistently saw the afflicted not as heretics but as the victims of diabolical possession or divine curse, and treated them accordingly. The dancers were subject to exorcisms or sent on pilgrimages. Never were they hauled before the inquisition.

Other authors have sought a chemical or biological origin for the dancing mania, and the chief contender has been ergot, a mould that grows on the stalks of damp rye. While seductively simple, this hypothesis is untenable. The chemicals contained in ergot do not allow for sustained dancing. They can certainly trigger violent convulsions and delusions, but not coordinated movements that last for days. Yet while the dancers were free from ergot, they almost certainly were delirious. Only in an altered state of consciousness could they have tolerated such extreme fatigue and the searing pain of sore, swollen and bleeding feet. Moreover, witnesses consistently spoke of the victims as being entranced, seeing terrifying visions and behaving with wild, crazy abandon. So what could have plunged hundreds of people into trances so deep that remorseless dancing became possible? Psychologists, neurologists and anthropologists have identified severe psychological distress as a factor increasing the likelihood of an individual entering an altered state. It is unlikely to be a coincidence, therefore, that in the year 1518 many people in Strasbourg were experiencing truly exceptional levels of hunger and mental anguish.

In a spin: the mysterious dancing epidemic of 1518. Endeavour. 2008 32(3): 117-121. doi: 10.1016/j.endeavour.2008.05.001

Ectomycorrhizal (ECM) fungi form a mutualistic symbiosis with tree roots and play key roles in forest ecosystems. In return for receiving nutrients and water from the soil via the roots, they receive carbohydrates as photosynthate from their host plants. As is the case for other soil fungal species, the composition of the ECM community is affected by both biotic and abiotic factors; these include climate changes, seasons, soil micro-site heterogeneity, soil and litter quality, host tree species and forest management. To describe in more detail the impact of environmental factors on community composition, long-term, year-round monitoring and a detailed spatial description of the community has to be carried out. However, analyses are very often hindered by a limited sample number and by the ephemeral or cryptic lifestyle of the fungi.

Over the last fifteen years, PCR-based molecular methods and DNA sequencing of nuclear and mitochondrial ribosomal DNA have been used routinely to identify mycorrhizal fungi. However, these methods are timeconsuming and are limited in the number of samples that can be treated in a realistic time frame. With automated molecular genotyping techniques, appropriate DNA databases and a better knowledge of internal transcribed spacer (ITS) variability within fungal species, identification of fungal taxa in environmental samples can now be expanded from the aforementioned methods to high-throughput molecular diagnostic tools, such as phylochips. So far, DNA arrays have been mainly used for genome-wide transcription profiling, but also for the identification of bacterial species from complex environmental samples or for the identification of a few genera of pathogenic fungi and Oomycetes.

Phylochips may comprise up to several thousand probes that target phylogenetic marker genes, such as 16S rRNA in bacteria or the ITS region in fungi. Phylochips have several advantages over traditional approaches, including higher specificity, cost efficiency, rapid identification and detection of target organisms, and the high numbers of samples throughput; therefore, they are increasingly used for the detection of bacterial and pathogenic fungi. In the ECM fungal ecology field, the first application of ribosomal DNA arrays was to develop a specific phylochip (on nylon membranes) to detect Suilloid fungi. Recently, this approach has also been used for truffle identification. No previous study has reported the construction and application of an ECM fungal phylochip to detect a large number of ECM fungal species that belong to various genera from environmental samples. This paper reports the first application of a custom ribosomal ITS phylochip to describe the community composition of ECM fungi on roots. The phylochip carried specific oligonucleotides for 95 fungal species that belong to 25 ECM fungal genera. The specificity of the oligonucleotides was evaluated using ITS amplicons of known reference species. The method was then used to describe ECM fungal communities that were obtained from 30-year-old spruce and beech plantations. The phylochip approach should be an attractive method for routine, accurate and reproducible monitoring of fungal species on specific sites, in which a high sample throughput is required.

Development and validation of an oligonucleotide microarray to characterise ectomycorrhizal fungal communities. BMC Microbiology 2009, 9: 241 doi:10.1186/1471-2180-9-241
In forest ecosystems, communities of ectomycorrhizal fungi (ECM) are influenced by biotic and abiotic factors. To understand their underlying dynamics, ECM communities have been surveyed with ribosomal DNA-based sequencing methods. However, most identification methods are both time-consuming and limited by the number of samples that can be treated in a realistic time frame. As a result of ongoing implementation, the array technique has gained throughput capacity in terms of the number of samples and the capacity for parallel identification of several species. Thus far, although phylochips (microarrays that are used to detect species) have been mostly developed to trace bacterial communities or groups of specific fungi, no phylochip has been developed to carry oligonucleotides for several ectomycorrhizal species that belong to different genera. We have constructed a custom ribosomal DNA phylochip to identify ECM fungi. Specific oligonucleotide probes were targeted to the nuclear internal transcribed spacer (ITS) regions from 95 fungal species belonging to 21 ECM fungal genera. The phylochip was first validated using PCR amplicons of reference species. Ninety-nine percent of the tested oligonucleotides generated positive hybridisation signals with their corresponding amplicons. Cross-hybridisation was mainly restricted at the genus level, particularly for Cortinarius and Lactarius species. The phylochip was subsequently tested with environmental samples that were composed of ECM fungal DNA from spruce and beech plantation fungal communities. The results were in concordance with the ITS sequencing of morphotypes and the ITS clone library sequencing results that were obtained using the same PCR products. To overcome cross-hybridisation problems, specific filter and evaluation strategies that used spot signal intensity were applied. Evaluation of the phylochip by hybridising environmental samples confirmed the possible application of this technology for detecting and monitoring ectomycorrhizal fungi at specific sites in a routine and reproducible manner.

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Amphibians such as frogs and toads are being driven to extinction by an aquatic fungus. This microbe, commonly called Bd, is spreading rapidly around the world and contributing to the decline in the biodiversity of the animals. In this article in Microbiology Today (pdf) Matthew Fisher believes that if control measures are not implemented, one-third of amphibian species could disappear:

Amphibians became the most ancient class of land-dwelling vertebrates when, 360 million years ago, Ichthyostega first hauled itself onto what was then Greenland. Since then, the amphibia have diversified into over 6,300 species that not only settled all continents except Antarctica, but also survived the catastrophic extinction events that overwhelmed their sister group, the dinosaurs. However, longevity of species is no guarantee of their future success; modern-day amphibians are suffering rates of extinction that far exceed those of any other class of vertebrates, including mammals and birds. Nearly one-third of amphibian species are threatened. The question of why amphibians are becoming extinct at these accelerated rates has puzzled scientists for three decades. While it is now clear that we are heading for a new anthropocene mass-extinction event as a consequence of human-driven planetary degradation, it has not been clear why this should be affecting amphibians more than other taxa. Further, many amphibian declines and extinctions were observed to occur in pristine environments that are relatively untouched by humans, such as rainforests and montane systems. A clue to the mystery came about when scientists working in Central America noted that the declines in amphibian biodiversity appeared to be occurring in a wave-like manner, with the initial losses being observed in Costa Rica, then spreading southwards towards the Panama Canal at rates of up to 43 km per year. These patterns of decline were suggestive of an epidemic, spreading pathogen, and in 1997 an international team of scientists discovered a new organism that appeared to be associated with many previously ‘enigmatic’ amphibian extinctions in two regions: Central America and north-eastern Australia. In 1999, the mycologist Joyce Longcore formally described this organism as new species of aquatic fungus and named it Batrachochytrium dendrobatidis.

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