Rare Organisms Research Articles

Denoising the Denoisers: an independent evaluation of microbiome sequence error-correction approaches.

High-depth sequencing of universal marker genes such as the 16S rRNA gene is a common strategy to profile microbial communities. Traditionally, sequence reads are clustered into operational taxonomic units (OTUs) at a defined identity threshold to avoid sequencing errors generating spurious taxonomic units. However, there have been numerous bioinformatic packages recently released that attempt to correct sequencing errors to determine real biological sequences at single nucleotide resolution by generating amplicon sequence variants (ASVs). As more researchers begin to use high resolution ASVs, there is a need for an in-depth and unbiased comparison of these novel “denoising” pipelines. In this study, we conduct a thorough comparison of three of the most widely-used denoising packages (DADA2, UNOISE3, and Deblur) as well as an open-reference 97% OTU clustering pipeline on mock, soil, and host-associated communities. We found from the mock community analyses that although they produced similar microbial compositions based on relative abundance, the approaches identified vastly different numbers of ASVs that significantly impact alpha diversity metrics. Our analysis on real datasets using recommended settings for each denoising pipeline also showed that the three packages were consistent in their per-sample compositions, resulting in only minor differences based on weighted UniFrac and Bray–Curtis dissimilarity. DADA2 tended to find more ASVs than the other two denoising pipelines when analyzing both the real soil data and two other host-associated datasets, suggesting that it could be better at finding rare organisms, but at the expense of possible false positives. The open-reference OTU clustering approach identified considerably more OTUs in comparison to the number of ASVs from the denoising pipelines in all datasets tested. The three denoising approaches were significantly different in their run times, with UNOISE3 running greater than 1,200 and 15 times faster than DADA2 and Deblur, respectively. Our findings indicate that, although all pipelines result in similar general community structure, the number of ASVs/OTUs and resulting alpha-diversity metrics varies considerably and should be considered when attempting to identify rare organisms from possible background noise.

Taxonomic annotation errors incorrectly assign the family Pseudoalteromonadaceae to the order Vibrionales in Greengenes: implications for microbial community assessments.

Next-generation sequencing has provided powerful tools to conduct microbial ecology studies. Analysis of community composition relies on annotated databases of curated sequences to provide taxonomic assignments; however, these databases occasionally have errors with implications for downstream analyses. Systemic taxonomic errors were discovered in Greengenes database (v13_5 and 13_8) related to orders Vibrionales and Alteromonadales. These orders have family level annotations that were erroneous at least one taxonomic level, e.g., 100% of sequences assigned to the Pseudoalteromonadaceae family were placed improperly in Vibrionales (rather than Alteromonadales) and >20% of these sequences were indeed Vibrio spp. but were improperly assigned to the Pseudoalteromonadaceae family (rather than to Vibrionaceae). Use of this database is common; we identified 68 peer-reviewed papers since 2013 that likely included erroneous annotations specifically associated with Vibrionales and Pseudoalteromonadaceae, with 20 explicitly stating the incorrect taxonomy. Erroneous assignments using these specific versions of Greengenes can lead to incorrect conclusions, especially in marine systems where these taxa are commonly encountered as conditionally rare organisms and potential pathogens.

Hydrothermal chimneys host habitat-specific microbial communities: analogues for studying the possible impact of mining seafloor massive sulfide deposits

To assess the risk that mining of seafloor massive sulfides (SMS) from extinct hydrothermal vent environments has for changing the ecosystem irreversibly, we sampled SMS analogous habitats from the Kairei and the Pelagia vent fields along the Indian Ridge. In total 19.8 million 16S rRNA tags from 14 different sites were analyzed and the microbial communities were compared with each other and with publicly available data sets from other marine environments. The chimneys appear to provide habitats for microorganisms that are not found or only detectable in very low numbers in other marine habitats. The chimneys also host rare organisms and may function as a vital part of the ocean’s seed bank. Many of the reads from active and inactive chimney samples were clustered into OTUs, with low or no resemblance to known species. Since we are unaware of the chemical reactions catalyzed by these unknown organisms, the impact of this diversity loss and bio-geo-coupling is hard to predict. Given that chimney structures can be considered SMS analogues, removal of sulfide deposits from the seafloor in the Kairei and Pelagia fields will most likely alter microbial compositions and affect element cycling in the benthic regions and probably beyond.

Abstract PCCLB-21: HEPCIDIN DEFICIENCY AND IRON OVERLOAD POTENTLY PROMOTE E. COLI SEPSIS IN MOUSE MODELS BUT THE HEPCIDIN AGONIST PR-73 IS PROTECTIVE

Aims & Objectives: We previously reported that the virulence in mice model of siderophilic (iron-loving) bacteria Vibrio vulnificus and Yersinia enterocolitica was potently enhanced by hepcidin deficiency and the resulting iron overload. Administration of hepcidin agonist PR-73 protected against lethal infections with these clinically rare organisms. To determine whether these findings are generalizable, we examined the role of hepcidin and iv iron in the pathogenesis of E. coli sepsis in mice. Methods Unselected E. coli blood isolates from children with sepsis were injected intraperitoneally into mice with genetic (hepcidin knockout mice, HKO) or iatrogenic (IV iron) iron overload and outcomes compared to control mice with normal iron status. We assessed mortality, bacterial burden and iron concentrations in blood and organs, and the response of infected mice to treatment with PR-73. Results Both genetic and iatrogenic iron overload potentiated infection with 8/10 E. coli isolates: iron-loaded mice developed sepsis with mortality of 60–100% within 24h while control mice suffered no mortality. 2 isolates manifested low virulence in mice, regardless of iron status. Treatment of infected HKO mice with two doses of PR-73 lowered serum iron and completely prevented sepsis-related mortality. The protective effect of hepcidin agonist PR-73 was attributable to the clearance of non-transferrin bound iron from plasma. Conclusions Hepcidin-induced hypoferremia not only protects against lethal sepsis from classical siderophilic pathogens but also from most clinical E. coli sepsis isolates. Hepcidin deficiency and iron overload are important and potentially common cofactors in susceptibility to sepsis. Hepcidin agonists may prove useful in early treatment of patients with sepsis.

Case Report: A rare case of prosthetic valve infective endocarditis caused by Aerococcus urinae.

Infective endocarditis (IE) is a serious and life-threatening cardiac condition, most commonly caused by staphylococci, Streptococcus viridans, and enterococci. However, in special settings, IE can be caused by rare organisms. Here we present a case of IE caused by Aerococcus urinae in a 75-year-old man with a bioprosthetic aortic valve. Aerococcusurinae is a gram-positive, catalase-negative microorganism and is usually an isolate of complicated urinary tract infections in the elderly male population. Improvements in diagnostic testing including use of matrix-assisted laser desorption ionization- a time of flight mass spectrometry (MALDI-TOF MS) have played an important role in recognition of Aerococcus urinae.

Predicting distributions, habitat preferences and associated conservation implications for a genus of rare fishes, seahorses (Hippocampus spp.)

AbstractAimTo identify useful sources of species data and appropriate habitat variables for species distribution modelling on rare species, with seahorses as an example, deriving ecological knowledge and spatially explicit maps to advance global seahorse conservation.LocationThe shallow seas.MethodsWe applied a typical species distribution model (SDM), maximum entropy, to examine the utility of (1) two versions of habitat variables (habitat occurrences vs. proximity to habitats) and (2) three sources of species data: quality research‐grade (RG) data, quality‐unknown citizen science (CS) and museum‐collection (MC) data. We used the best combinations of species data and habitat variables to predict distributions and estimate species–habitat relations and threatened status for seahorse species.ResultsWe demonstrated that using “proximity to habitats” and integrating all species datasets (RG, CS and MC) derived models with the highest accuracies among all dataset variations. Based on this finding, we derived reliable models for 33 species. Our models suggested that only 0.4% of potential seahorse range was suitable to more than three species together; seahorse biogeographic epicentres were mainly in the Philippines; and proximity to sponges was an important habitat variable. We found that 12 “Data Deficient” species might be threatened based on our predictions according to IUCN criteria.Main conclusionsWe highlight that using proper habitat variables (e.g., proximity to habitats) is critical to determine distributions and key habitats for low‐mobility animals; collating and integrating quality‐unknown occurrences (e.g., CS and MC) with quality research data are meaningful for building SDMs for rare species. We encourage the application of SDMs to estimate area of occupancy for rare organisms to facilitate their conservation status assessment.

Testate amoebae community analysis as a tool to assess biological impacts of peatland use

As most ecosystems, peatlands have been heavily exploited for different human purposes. For example, in Finland the majority is under forestry, agriculture or peat mining use. Peatlands play an important role in carbon storage, water cycle, and are a unique habitat for rare organisms. Such properties highlight their environmental importance and the need for their restoration. To monitor the success of peatland restoration sensitive indicators are needed. Here we test whether testate amoebae can be used as a reliable bioindicator for assessing peatland condition. To qualify as reliable indicators, responses in testate amoebae community structure to ecological changes must be stronger than random spatial and temporal variation. In this study, we simultaneously assessed differences between the effects of seasonality, intermediate scale spatial variation and land uses on living testate amoebae assemblages in natural, forested and restored peatlands. We expected the effects of seasonality on testate amoebae communities to be less pronounced than those of land use and within site variation. On average, natural sites harboured the highest richness and density, while the lowest numbers were found at forestry sites. Despite small changes observed in taxa dominance and differences in TA community structure between seasons and years at some sites, spatial heterogeneity, temperature, pH, nor water table depth seemed to significantly affect testate amoebae communities. Instead, observed differences were related to type of land use, which explained 75% of the community variation. Our results showed that testate amoebae community monitoring is a useful tool to evaluate impacts of human land use on boreal peatlands.

Linking Associations of Rare Low-Abundance Species to Their Environments by Association Networks.

Studies of microbial communities by targeted sequencing of rRNA genes lead to recovering numerous rare low-abundance taxa with unknown biological roles. We propose to study associations of such rare organisms with their environments by a computational framework based on transformation of the data into qualitative variables. Namely, we analyze the sparse table of putative species or OTUs (operational taxonomic units) and samples generated in such studies, also known as an OTU table, by collecting statistics on co-occurrences of the species and on shared species richness across samples. Based on the statistics we built two association networks, of the rare putative species and of the samples respectively, using a known computational technique, Association networks (Anets) developed for analysis of qualitative data. Clusters of samples and clusters of OTUs are then integrated and combined with metadata of the study to produce a map of associated putative species in their environments. We tested and validated the framework on two types of microbiomes, of human body sites and that of the Populus tree root systems. We show that in both studies the associations of OTUs can separate samples according to environmental or physiological characteristics of the studied systems.

Hand infections

The integument provides only a limited barrier to protect the multiple functional structures of the hand from infection by common bacteria and other rare organisms. If infection is limited to just skin cellulitis, then antibiotics and careful monitoring may be all that is needed. Once bacteria become trapped in a deeper soft-tissue space and form an abscess then debridement must be performed to evacuate the purulence and necrotic tissue. Specialized compartments in the hand such as the pulp space, nail folds, joint cavities, and tendon sheaths facilitate bacterial containment and abscess formation, rendering immune response less effective. Osteomyelitis can originate from direct penetration or spread from an adjacent soft-tissue infection. Orthopaedic devices foster the development of a biofilm that can harbor bacteria, making resolution more difficult. The surgeon should obtain tissue for laboratory analysis at the deep infection site and subsequently administer empiric antibiotics while awaiting culture results. Greater duration of antibiotics is needed for infections of synovial tissues and bone. Unique infections caused by rare organisms may affect the hand, requiring recognition based on specific clinical features and confirmation by specialized laboratory testing.

Case Report: A rare case of prosthetic valve infective endocarditis caused by Aerococcus urinae

Infective endocarditis (IE) is a serious and life-threatening cardiac condition, most commonly caused by staphylococci, Streptococcus viridans, and enterococci. However, in special settings, IE can be caused by rare organisms. Here we present a case of IE caused by Aerococcus urinae in a 75-year-old man with a bioprosthetic aortic valve. Aerococcus urinae is a gram-positive, catalase-negative microorganism and is usually an isolate of complicated urinary tract infections in the elderly male population. . Improvements in diagnostic testing including use of matrix-assisted laser desorption ionization– a time of flight mass spectrometry (MALDI-TOF MS) have played an important role in isolating Aerococcus.

Improving the ecological relevance of aquatic bacterial communities in biodegradability screening assessments

Concentrating cells from aqueous samples is a common requirement for the enumeration of biomass, investigations of microbial diversity and detection of relatively rare organisms in the environment. Accurately representing the initial sampled environments in the concentrated cells is of particular importance when the subsequent analyses have tangible environmental, economic and societal consequences, as is the case with environmental exposure and risk assessment of chemicals. This study investigated the potential use of four different cell concentration methods: centrifugation, membrane filtration, tangential flow filtration and column colonisation. These methods were assessed against a series of scientific and practical criteria, including: similarity of concentrated community to initial environmental sample; cell concentration achieved; biodegradation test outcome; sample throughput; and capital and maintenance costs. All methods increased cell concentration by as little as 10-fold to as much as 1000-fold. DGGE and 454 pyrosequencing analysis showed concentrated communities to have >60% similarity to each other, and the initial sample. There was a general trend for a more reliable assessment of 4-nitrophenol biodegradation in 96-well plate biodegradation assays, with increasing cell concentration. Based on the selection criteria, it is recommended that there is not one concentration method fit for all purposes, rather, the appropriate method should be selected on a case-by-case basis. Membrane filtration would be the most suitable method for low sample volumes; the increased throughput capacity of tangential flow filtration renders it most suitable for large volumes; and centrifugation is most suitable for samples with high initial biomass concentrations. The poor similarity in microbial community composition of the column colonised samples compared to the initial samples, suggested a concentration basis; this combined with its low sample throughput precluded this approach for future concentration studies of planktonic bacterial samples.

Living quarters of a living fossil-Uncovering the current distribution pattern of the rediscovered Hula painted frog (Latonia nigriventer) using environmental DNA.

One of the greatest challenges of effective conservation measures is the correct identification of sites where rare and elusive organisms reside. The recently rediscovered Hula painted frog (Latonia nigriventer) has not been seen for many decades and was therefore categorized extinct. Since its rediscovery in 2011, individuals from the critically endangered species have been found, with great effort, only in four restricted sites. We applied the environmental DNA (eDNA) approach to search for new populations of the Hula painted frog in suitable aquatic habitats. We further used the eDNA data to classify the landscape factors associated with the species distribution and to predict its suitable habitats. We sampled 52 aquatic sites in the Hula Valley during the spring of 2015 and 2016 and amplified the samples with a species-specific qPCR assay. DNA of the Hula painted frog was detected in 22 of the sites, all of which clustered within three main areas. A boosting classification model showed that soil type, vegetation cover and the current and former habitats are all key predictors of the frog's current distribution. Intriguingly, the habitat suitability models reveal a high affinity of the species to its long-lost habitat of the historical wetlands. Our findings encourage a series of informed searches for new populations of this threatened frog and provide guidance for future conservation management programmes. In the era of global conservation crisis of amphibians, developing the eDNA approach, a reliable detection method for many critically endangered and elusive amphibians, is of particular importance.

White Cart Loom

Abstract British artists Vicky Isley and Paul Smith (collectively known as boredomresearch) were commissioned in 2016 by the University of the West of Scotland (UWS) to produce a new digital artwork with the aim of supporting Paisley’s bid to be City of Culture 2021. The commission was co-funded by UWS and Renfrewshire Council, Scotland. The resulting artwork titled White Cart Loom weaves a narrative combining Paisley’s rich industrial past with current scientific and ecological work, fighting to save a rare organism from extinction.

First case report of Providencia Rettgeri neonatal sepsis

BackgroundProvidencia are gram negative motile rods and is a member of the Enterobacteriaceae family. It consists of five species, namely Providencia alcalifaciens, Providencia rustigianii, Providencia stuartii, Providencia rettgeri and Providencia heimbachae. These are opportunistic pathogens and leads to infections in immunocompromised host. Providencia rettgeri has been associated with the nosocomial infections of the urinary tract and infections of wounds, burns and blood. Providencia rettgeri is very rare cause of neonatal sepsis and we report first case of neonatal late onset sepsis secondary to it.Case presentationA term male infant presented on day 4 of post-natal life with the complaint of decreased appetite, fast respiration and lethargy. The clinical examination showed features of sepsis and shock with chest radiogram showing pneumonia. The infant was started on invasive ventilation, intravenous fluids, antibiotic and inotropes. The blood culture was suggestive of multi-drug resistant P. rettgeri. The antibiotics were changed according to organism antibiotic susceptibility pattern and infant gradually improved and was discharged successfully.ConclusionProvidencia rettgeri is a very rare organism to cause neonatal sepsis. The management involves early diagnosis, treatment with appropriate antibiotics and finding the source of infection.

Peritoneal dialysis-associated catheter infection caused by Mycobacterium abscessus in an elderly patient who was successfully treated with catheter removal.

An 89-year-old Japanese man on peritoneal dialysis (PD) was suspected of having a PD-associated catheter infection. He visited the hospital because of the discharge of pus from the exit site of his catheter. Gram staining of the pus showed Gram-positive bacilli, but these were acid-fast bacilli. The rapidly growing nontuberculous mycobacteria, Mycobacterium abscessus, was isolated. PD catheter removal and debridement were immediately performed. The patient received combination antibiotic therapy. His clinical course was good, but he required hemodialysis due to the discontinuation of PD. However, the patient and his family chose not to continue hemodialysis even when the symptoms of uremia appeared. Best supportive care was arranged by his primary care physician. M. abscessus is a rare causative organism for PD-associated catheter infections and is difficult to treat. In our case, a rapid and precise diagnosis was made using acid-fast staining and Mycobacterium culture. The risk of nontuberculous mycobacterial infections should be considered in patients on PD.

First report of neonatal bacteraemia caused by blaNDM-1 Raoultella ornithinolytica

Raoultella ornitholytica, an environmental organism, has only recently been reported as a cause of infections in humans, and is associated mainly with bacteraemia in elderly patients. Carbapenem resistance is unusual in Raoultella spp. We describe the first paediatric case of blaNDM-1 Raoultella ornitholytica bacteraemia in a preterm neonate that developed three days after hospitalisation. This case report discusses a rare organism that had an unexpected susceptibility profile and caused bacteraemia in a neonate. Treatment options are explored.

Panfungal Polymerase Chain Reaction for Identification of Fungal Pathogens in Formalin-Fixed Animal Tissues

Identification of fungal organisms often poses a problem for pathologists because the histomorphology of some fungal organisms is not specific, fresh tissues may not be available, and isolation and identification in culture may take a long time. The purpose of this study was to validate the use of panfungal polymerase chain reaction (PCR) to identify fungal organisms from formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed paraffin-embedded curls were tested from 128 blocks containing canine, feline, equine, and bovine tissues with cutaneous, nasal, pulmonary, and systemic fungal infections, identified by the presence of fungi in histologic sections. Quantitative scoring of histologic sections identified rare (11.9%), occasional (17.5%), moderate (17.5%), or abundant (53.1%) fungal organisms. DNA was isolated from FFPE tissues and PCR was performed targeting the internal transcribed spacer 2 (ITS-2) region, a segment of noncoding DNA found in all eukaryotes. Polymerase chain reaction products were sequenced and identified at ≥97% identity match using the Basic Local Alignment Search Tool and the NCBI database of ITS sequences. Of the 128 blocks, 117 (91.4%) yielded PCR products and high-quality sequences were derived from 89 (69.5%). Sequence and histologic identifications matched in 79 blocks (61.7%). This assay was capable of providing genus- and species-level identification when histopathology could not and, thus, is a beneficial complementary tool for diagnosis of fungal diseases.

Perspectives on the Controlled Propagation, Augmentation, and Reintroduction of Freshwater Mussels (Mollusca: Bivalvia: Unionoida)

Controlled propagation, augmentation, and reintroduction (PAR) of rare and endangered aquatic organisms has become a priority action for recovery and delisting, and in many cases is an action of “last resort” to either restore or maintain existing populations. The guiding principle of PAR efforts should be to avoid harming existing populations of congeneric or nontarget species and also minimize risks to extant populations and habitats. Controlled PAR of freshwater mussels should not be a long-term management strategy conducted in perpetuity and should not be used as a substitute for recovery tasks such as habitat restoration or addressing the causes of endangerment. The determination to pursue controlled PAR for freshwater mussels should follow a thorough evaluation of the status of existing wild populations, an agreement that PAR in the historic range is needed, and a conclusion that suitable habitat for long-term success is present. The primary purpose of any efforts to augment or reintroduce animals should be to establish free-ranging wild populations. Concomitant with this goal is the distinct possibility that these activities can represent appreciable genetic or ecological risks to resident animals, both nontarget taxa and wild conspecifics. To maintain the integrity of the fauna, communities, and ecosystems it is imperative that these risks be carefully considered before conducting controlled PAR. In this paper we pose several questions that we believe are important to consider before initiating PAR of freshwater mussels. We also recommend actions, some already used at individual facilities or by agencies, that we believe will aid in developing a more uniform approach to controlled PAR and safeguarding the ecological and genetic integrity of freshwater mussel communities.

An Efficient Antioxidant System in a Long-Lived Termite Queen.

The trade-off between reproduction and longevity is known in wide variety of animals. Social insect queens are rare organisms that can achieve a long lifespan without sacrificing fecundity. The extended longevity of social insect queens, which contradicts the trade-off, has attracted much attention because it implies the existence of an extraordinary anti-aging mechanism. Here, we show that queens of the termite Reticulitermes speratus incur significantly lower oxidative damage to DNA, protein and lipid and have higher activity of antioxidant enzymes than non-reproductive individuals (workers and soldiers). The levels of 8-hydroxy-2’-deoxyguanosine (oxidative damage marker of DNA) were lower in queens than in workers after UV irradiation. Queens also showed lower levels of protein carbonyls and malondialdehyde (oxidative damage markers of protein and lipid, respectively). The antioxidant enzymes of insects are generally composed of catalase (CAT) and peroxiredoxin (Prx). Queens showed more than two times higher CAT activity and more than seven times higher expression levels of the CAT gene RsCAT1 than workers. The CAT activity of termite queens was also markedly higher in comparison with other solitary insects and the queens of eusocial Hymenoptera. In addition, queens showed higher expression levels of the Prx gene RsPRX6. These results suggested that this efficient antioxidant system can partly explain why termite queens achieve long life. This study provides important insights into the evolutionary linkage of reproductive division of labor and the development of queens’ oxidative stress resistance in social insects.

Clinical implications of Paracoccus yeeii bacteremia in a patient with decompensated cirrhosis

Infections in patients with cirrhosis are common among those who develop variceal hemorrhage. Prophylactic antimicrobial treatment with third generation cephalosporins is recommended in patients with advanced cirrhosis and gastrointestinal hemorrhage. However no infectious source is identified in up to 50% of patients with cirrhosis and clinical sepsis. We report the first case of Paracoccus yeeii bacteremia in a patient with decompensated cirrhosis who presented with variceal hemorrhage. This rare gram negative organism that occurs naturally in the soil has been difficult to isolate until recent technological advances and may not be susceptible to third generation cephalosporins. Our case reinforces the challenges in isolating rare infections in patients with cirrhosis, the need to consider uncommon organisms in infected but culture negative patients with cirrhosis, and the importance of optimizing antimicrobials to reduce the incidence of drug resistant organisms.