Single Microorganism Research Articles

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Introduction When we were invited by the Health Informatics Society of Australia (HISA) in March 2012 to participate in the Scientific Program Committee of a new conference on big data, we had mixed feelings. On one hand, it seemed very exciting to develop this theme in a very appropriate time and try to address aspects which are highly relevant for clinical and biomedical sciences. However, at the same time, it seemed a little bit bold to start this new activity when the concept of big data was still poorly defined and did not yet exist too many (reported) experiences in Australia. Fortunately, we had the precedent of a successful Data Governance conference series and we must say that during the time worked on the organisation of the event, things were on our side. Today there is a greater awareness of the importance of the concept of big data science and many centres are developing projects in this area. Thanks to the agreement with the journal BMC Health Information Science and Systems (BMC HISS) we have the pleasure to present in this special issue the best papers submitted to this conference. The articles collected in this special issue show the diversity of applications and definitions of big data in health and have been organised around biological levels of increasing complexity from molecules to organisms and from patients to social networks. The process leading to the inclusion of the papers in this special issue started during the review of the submissions for the conference, where three reviewers evaluated the submissions and invited seven submisions to be extended into a full article to be included in this special issue. These submitted full papers underwent a new round of reviews before their acceptance for publication in this special issue. A first set of papers was focused in the relevance and application of big data at what would be defined as the lower biological complexity levels, from molecules to microorganisms. In their paper B. Goudey et al focused in the study of single nucleotide variants at the molecular level and the computing requirements to deal with this source of big data. Expanding the analysis from single nucleotides to genomes and their application in public health L. I. Rusu et al presented work showing the increasing importance of the use of next generation sequencing in microbiology and how these data can be considered big data. Continuing in the microbiology area, G. Lopez-Campos et al focused their work on the relevance of big data in the study of single microorganisms not only based on the amount but also in the variety of data that have to be managed nowadays for their characterisation and study. The rest of the selected articles are related with patient data at different levels. The paper by T.D. Nguyen et al relates big data with medical imaging presenting all the steps required to develop and run a multimodality research imaging informatics repository. Another source of big data considered by B. Gallego et al are hospital electronic data collected routinely in hospitals that can be analysed to improve patient safety. Another area covered in this issue is the relevance of big data associated with the concept of self-quantification, in their work M. Almaki et al presented some of the challenges that this approach is posing and its relationship with big data concepts. Finally, and from a social perspective G. Zuccon et al addressed the feasibility of using big data available through social networks and other web 2.0 tools such as Twitter to detect and report influenza like diseases as well as the validity of these approaches. All these articles represent only the tip of the iceberg of the use of big data for health applications. We consider that it is worth to remark the diversity of applications of the works collected in this issue and how they show the

ArchaeProfile: A Database of Archaea and their Origins of Replication

Archaea are single cell microorganisms having several unique characteristics which differentiate them from bacteria. One of the key features which make archaea distinct from bacteria is the replication process, which is very different and resembles that of the eukaryotes. In-vivo mapping of the ori site in Archae is a time consuming and tedious job due to complexity involved in the culture of archaeal colony, which puts challenges as well as opportunity to scientist to devise in-silico method to map the Ori site in archaeal genomes. Z-curve approach is a widely used insilico method to predict the Ori site in archaea, but it is not equally successful for all archaeal genomes. Several other parameters like copy number and location of the cdc6 gene, AT rich region with the presence of origin recognition boxes (ORB) provide a better estimate of the Ori site in archaea. The motivation behind development of Archae Profile database is to predict the location and the number of putative Ori sites in archaeal genomes based on purinepyrimidine ( R-Y) and amino-keto(M-K) disparity curve along with the consensus ORB sequences, cdc6 gene copy number, their location and upstream AT richness. Quick update cycle and easy browser interface makes Archae Profile distinct from other databases. Another important feature is the integration of tools for plotting disparity plot of a given genome sequence and finding specific repeats with copy number and location in a sequence. ArchaeProfile will be updated timely and the emphasis will be to integrate other tools for genome analysis as well as new search features in the database. Presently Archaea Profile has Ori related data of 122 archeal genome which is likely to increase with time. Availability: ArchaeProfile can be accessed freely from http://www.bioinfommv.in/archaeprofile. Data available on the database could be used for further analysis and tutorial purpose.

Challenging biological limits with microfluidic single cell analysis.

Challenging biological limits with microfluidic single cell analysis.

Role of the intestinal microbiota and fecal transplantation in inflammatory bowel diseases.

Ulcerative colitis and Crohn's disease are the two major types of inflammatory bowel disease (IBD). Despite intensive study, it is still challenging because the precise etiology and pathogenesis remains unclear. Studies have shown that IBD is associated with changes in the composition of intestinal microbiota, as either a cause or a consequence of abnormal host immune response in genetic susceptible population. Two specific microorganisms (Mycobacterium avium subsp. paratuberculosis and Escherichia coli) get more widely studied, but till now no single microorganism has been identified as the only pathogen. Genetic susceptibility data also suggest impaired handling of bacteria as well as an improper immune response to potential pathogens. The microbiota provides new therapeutic methods, and fecal microbiota transplantation may restore the balance of intestinal flora to supplement or optimize the current therapies.

Expression of a manganese peroxidase isozyme 2 transgene in the ethanologenic white rot fungus Phlebia sp. strain MG-60

BackgroundThe white-rot fungus Phlebia sp. strain MG-60 was proposed as a candidate for integrated fungal fermentation process (IFFP), which unifies aerobic delignification and semi-aerobic consolidated biological processing by a single microorganism based on its ability to efficiently degrade lignin and ferment the sugars from cellulose. To improve IFFP, the development of a molecular breeding method for strain MG-60 is necessary. The purpose of this study is to establish the transformation method for the strain MG-60 and to obtain the over-expressing transformants of lignin-degrading enzyme, manganese peroxidase.FindingsIn the present study, the expression vector regulated by Phlebia brevispora glyceraldehyde-3-phosphate dehydrogenase promoter and terminator was constructed. A polyethylene glycol transformation method for the ethanol-fermenting white-rot fungus Phlebia sp. MG-60 was established with high transformation efficiency, and the manganese peroxidase isozyme 2 gene (MGmnp2) transformants were obtained, showing higher MnP activity than control transformants. MGmnp2 transformants showed higher selective lignin degradation on Quercus wood powder.ConclusionsThis first report of MG-60 transformation provides a useful methodology for widely accessible to interested researches. These results indicate the possibility of metabolic engineering of strain MG-60 for improving IFFP.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-699) contains supplementary material, which is available to authorized users.

Aerobic and sequential anaerobic fermentation to produce xylitol and ethanol using non-detoxified acid pretreated corncob.

BackgroundFor economical bioethanol production from lignocellulosic materials, the major technical challenges to lower the production cost are as follows: (1) The microorganism should use efficiently all glucose and xylose in the lignocellulose hydrolysate. (2) The microorganism should have high tolerance to the inhibitors present in the lignocellulose hydrolysate. The aim of the present work was to combine inhibitor degradation, xylitol fermentation, and ethanol production using a single yeast strain.ResultsA new process of integrated aerobic xylitol production and anaerobic ethanol fermentation using non-detoxified acid pretreated corncob by Candida tropicalis W103 was proposed. C. tropicalis W103 is able to degrade acetate, furfural, and 5-hydromethylfurfural and metabolite xylose to xylitol under aerobic conditions, and the aerobic fermentation residue was used as the substrate for ethanol production by anaerobic simultaneous saccharification and fermentation. With 20% substrate loading, furfural and 5-hydroxymethylfurfural were degraded totally after 60 h aerobic incubation. A maximal xylitol concentration of 17.1 g l-1 was obtained with a yield of 0.32 g g-1 xylose. Then under anaerobic conditions with the addition of cellulase, 25.3 g l-1 ethanol was produced after 72 h anaerobic fermentation, corresponding to 82% of the theoretical yield.ConclusionsXylitol and ethanol were produced in Candida tropicalis W103 using dual-phase fermentations, which comprise a changing from aerobic conditions (inhibitor degradation and xylitol production) to anaerobic simultaneous saccharification and ethanol fermentation. This is the first report of integrated xylitol and ethanol production from non-detoxified acid pretreated corncob using a single microorganism.

Epidemiology and Prevalence of Bloodstream Infections in a Regional Hospital in Northern Taiwan During 2008–2013

Background Diagnosing bloodstream infections (BSIs) is a critical function of clinical microbiology laboratories. To reveal the prevalence of BSIs in a hospital in Northern Taiwan, which was established in July 2008, we investigated the clinical characteristics of patients with positive blood cultures from July 2008 to December 2013. Methods The medical records of the patients were retrospectively reviewed. In total, 104,641 blood culture sets were collected and analyzed. Results Microorganisms grew on 10.28% of these sets, as follows: 5.48% exhibited growth of a single microorganism; 1.56% exhibited growth of more than one microorganism; and 3.24% exhibited growth of contaminants. Furthermore, 5739 monomicrobial isolates included: Gram-positive cocci (22.02%); Enterobacteriaceae (56.51%); glucose nonfermentative Gram-negative bacteria (7.27%); yeast (6.22%); and anaerobic bacteria (6.64%). Most microorganisms were identified as follows: Escherichia coli (33.80%); Staphylococcus aureus (14.20%); Klebsiella pneumoniae (11.41%); Pseudomonas aeruginosa (3.17%); and the Acinetobacter calcoaceticus–Acinetobacter baumannii (Acb) complex (2.68%). Furthermore, among infants aged ≤3 months, Group B Streptococcus (GBS) and S. aureus were the leading pathogens causing bacteremia, whereas among infants 3–12 months old and children 1–4 years old, Salmonella species were the leading pathogens causing bacteremia. The prevalence of Streptococcus pneumoniae increased from the 3–12-month-old age group and reached a peak in the 5–12-year-old age group. For patients aged >13 years, the most common pathogens were E. coli, S. aureus, and K. pneumoniae. Conclusion We propose that the types and prevalence of BSIs vary according to age group and exhibit substantial geographical differences.

Succinate production from xylose‐glucose mixtures using a consortium of engineered Escherichia coli

The conversion of variable sugar mixtures into biochemicals poses a challenge for a single microorganism. For example, succinate has not been effectively generated from mixtures of glucose and xylose. In this work, a consortium of two Escherichia coli strains converted xylose and glucose to succinate in a dual phase aerobic/anaerobic process. First, the optimal pathway from xylose or glucose to succinate was determined by expressing either heterologous pyruvate carboxylase or heterologous adenosine triphosphate‐forming phosphoenol pyruvate (PEP) carboxykinase. Expression of PEP carboxykinase (pck) resulted in higher yield (0.86 g/g) and specific productivity (155 mg/gh) for xylose conversion, while expression of pyruvate carboxylase (pyc) resulted in higher productivity (76 mg/gh) for glucose conversion. Then, processes using consortia of the two optimal xylose‐selective and glucose‐selective strains were designed for two different feed ratios of glucose/xylose. In each case the consortia generated over 40 g/L succinate efficiently with yields greater than 0.90 g succinate/g total sugar. This study demonstrates two advantages of microbial consortia for the conversion of sugar mixtures: each sugar‐to‐product pathway can be optimized independently, and the volumetric consumption rate for each sugar can be controlled independently, for example, by altering the biomass concentration of each consortium member strain.

Effect of Saccharomyces cerevisiae and Zymomonas mobilis on the co-fermentation of sweet sorghum bagasse hydrolysates pretreated under varying conditions

Hydrolysates from sweet sorghum bagasse pretreatment normally contains hexose and pentose sugars, and this complex mixture of sugars presents a challenge for a single microorganism to effectively ferment all sugars to ethanol. In this study, synergistic effects on the co-fermentation of the hydrolysates using Sacchromyces cerevisiae and Zymomonas mobilis ATCC31825 at different ratios were studied. An inoculum of mixed cultures (1:3 and 5:10 g/L, Z. mobilis to S. cerevisiae ratios) was investigated. Each mixed culture was added to the hydrolysates at pH 4.8 and incubated 32 °C for 24 h. The mixture of Z. mobilis to S. cerevisiae at 5:10 g/L showed the highest synergistic effect with ethanol yields of 0.5 g/g. Since the yield for co-culture was significantly higher than the sum of yields from each microorganism, the improvements can be directly related to co-fermentation of hydrolysate by S. cerevisiae and Z. mobilis.

Antibiotic therapy for diabetic foot infections in a tertiary care hospital in Jakarta, Indonesia

AimsThe purpose of this study was to determine the microbiology of diabetic foot infections and to assess the antibiotic susceptibility patterns. Materials and methodsA cross sectional retrospective study of 35 patients with diabetic foot infections hospitalized at the internal medicine clinic of Dr. Mintohardjo Navy Hospital, Jakarta. The data were collected from patient medical records retrospectively. The classification of the diabetic foot infections was evaluated according to Meggit–Wagner's Classification. Identification of causative microorganisms was performed by standard microbiologic methods. Antibiotic susceptibility testing was performed using disk-diffusion method. ResultsDuring January to December 2012, a total of 288 of diabetic patients were admitted to hospital, and 35 patients had diabetic foot infections. According to Meggit–Wagner's classification the most common disease was grade 3 in 31.4% patients, followed by grade 2 in 25.7%, and grade 4 in 17.3% of patients. Conservative diabetic control care was carried out in 37.1% of patients, and surgical intervention was carried out in 62.9% of patients. A total of 59 pathogens were identified. The most common infecting microorganism isolated on pus cultures was Staphylococcus aureus (47.5%), followed by Pseudomonas spp (16.9%), E. coli (10.2%), Streptococcus spp. (8.5%), Enterobacter spp. (7.0%), Proteus spp. (6.7%), and Acinetobacter spp. (3.2%). Overall, 37.2% of the diabetic foot infection caused by a single microorganism, and 62.8% had polymicrobial infections. The most frequently administrated antibiotic was ceftriaxone (40.0%), followed by ciprofloxacin (11.4%), and meropenem (8.6%). ConclusionDiabetic foot infections (62.8%) were polymicrobial. S. aureus was most commonly found in the foot infection. Most of the microorganisms isolated from diabetic foot infection were resistant to many types of antibiotics.

Central Venous Catheter Salvage in Home Parenteral Nutrition Catheter-Related Bloodstream Infections: Long-Term Safety and Efficacy Data.

Catheter-related bloodstream infections (CRBSIs) are a serious complication in the provision of home parenteral nutrition (HPN). Antibiotic salvage of central venous catheters (CVCs) in CRBSI is recommended; however, this is based on limited reports. We assessed the efficacy of antibiotic salvage of CRBSIs in HPN patients. All confirmed CRBSIs occurring in patients receiving HPN in a national intestinal failure unit (IFU), between 1993 and 2011, were analyzed. A standardized protocol involving antibiotic and urokinase CVC locks and systemic antibiotics was used. In total, 588 patients were identified with a total of 2134 HPN years, and 297 CRBSIs occurred in 137 patients (65 single and 72 multiple CRBSIs). The overall rate of CRBSI in all patients was 0.38 per 1000 catheter days. Most (87.9%) infections were attributable to a single microorganism. In total, 72.5% (180/248) of CRBSIs were salvaged when attempted (coagulase-negative staphylococcus, 79.8% [103/129], Staphylococcus aureus, 56.7% [17/30]; polymicrobial infections, 67.7% [21/30]; and miscellaneous, 66.1% [39/59]). CVC salvage was not attempted in 49 episodes because of life-threatening sepsis (n = 18), fungal infection (n = 7), catheter problems (n = 20), and CVC tunnel infection (n = 4). Overall, the CVC was removed in 33.7% (100/297) of cases. There were 5 deaths in patients admitted to the IFU for management of the CRBSI (2 severe sepsis at presentation, 3 metastatic infection). This is the largest reported series of catheter salvage in CRBSIs and demonstrates successful catheter salvage in most cases when using a standardized protocol.

Isoprene production in Synechocystis under alkaline and saline growth conditions

Photosynthesis for the generation of isoprene in cyanobacteria was demonstrated with Synechocystis, entailing a process where a single host microorganism acts as both photocatalyst and processor, photosynthesizing and emitting isoprene hydrocarbons. A practical aspect of the commercial exploitation of this process in mass culture is the need to prevent invading microorganisms that might cause a culture to crash, and to provide an alternative to freshwater in scale-up applications. Growth media poised at alkaline pH are desirable in this respect, as high pH might favor the growth of the cyanobacteria, while at the same time discouraging the growth of invading predatory microbes and grazers. In addition, demonstration of salinity tolerance would enable the use of seawater for cyanobacteria cultivations. However, it is not known if Synechocystis growth and the isoprene-producing metabolism can be retained under such theoretically non-physiological conditions. We applied the gaseous/aqueous two-phase photobioreactor system with Synechocystis transformed with the isoprene synthase gene (SkIspS) of Pueraria montana (kudzu). Rates of growth and isoprene production are reported under control, and a combination of alkalinity and salinity conditions. The results showed that alkalinity and salinity do not exert a negative effect on either cell growth or isoprene production rate and yield in Synechocystis. The work points to a practical approach in the design of cyanobacterial growth media for applications in commercial scale-up and isoprene production.

Diabetic foot: infections and outcomes in Iranian admitted patients.

Background:Diabetes mellitus (along with its complications) has become a global problem. Diabetic foot infection, among the most common complications, is responsible for 40 to 50% of foot amputations. Antibiotic-resistant microorganisms, however, have compromised empiric therapy in the infected patients.Objectives:The current study aimed to determine the most common microorganisms involved in diabetic foot infection in order to minimize the failure of antibiotic therapy and the risk of developing complications.Patients and Methods:All patients with diabetic foot infection admitted to the infectious diseases, surgery and endocrinology wards of two teaching hospitals from 2007 to 2010 (n = 196) were recruited. In this retrospective study, demographic characteristics, type of lesions, history of hospitalization/antibiotic therapy, isolated microorganisms, clinical complications, administered treatment (medical or surgical) and outcome were recorded.Results:Patients’ mean age was 60.84 (± 10.30) years. Totally, 113 (57.65%) of the patients were male and 83 (42.35%) were female. According to Wagner’s grading, deep ulcers with/without osteomyelitis accounted for the majority of lesions. A single microorganism was isolated (most common: Escherichia coli, Staphylococcus aureus and Klebsiella spp.) from 81 of the patients (80.20%); while for the remaining polymicrobial infection was reported. Isolated pathogens showed no significant correlation with duration of diabetes, type of the lesions (P = 0.13) and history of hospitalization (P = 0.61). The majority of patients (n = 118, 60.20%) were treated surgically; however 11 patients expired due to sepsis. Amputation (most common at toes and below the knee) was performed for 89 patients (45.40%). The response rate to medical treatment was 31.6% for single-pathogen and 10% for polymicrobial infection (with a 30% mortality rate).Conclusions:Physicians are recommended to take microbiological cultures before starting empirical therapy recommended to cover Gram-negative microorganisms in order to lower the risk of antibiotic resistance.

Potential of fungal co-culturing for accelerated biodegradation of petroleum hydrocarbons in soil

The potential of fungal co-culture of the filamentous Pestalotiopsis sp. NG007 with four different basidiomycetes – Trametes versicolor U97, Pleurotus ostreatus PL1, Cerena sp. F0607, and Polyporus sp. S133 – for accelerating biodegradation of petroleum hydrocarbons (PHCs) was studied using three different physicochemical characteristic PHCs in soil. All the combinations showed a mutual intermingling mycelial interaction on the agar plates. However, only NG007/S133 (50/50) exhibited an optimum growth rate and enzymatic activities that supported the degradation of asphalt in soil. The co-culture also degraded all fractions at even higher concentrations of the different PHCs. In addition, asphaltene, which is a difficult fraction for a single microorganism to degrade, was markedly degraded by the co-culture, which indicated that the simultaneous biodegradation of aliphatic, aromatic, resin, and asphaltene fractions had occurred in the co-culture. An examination of in-vitro degradation by the crude enzymes and the retrieval fungal culture from the soil after the experiment confirmed the accelerated biodegradation due to enhanced enzyme activities in the co-culture. The addition of piperonyl butoxide or AgNO3 inhibited biodegradation by 81–99%, which demonstrated the important role of P450 monooxygenases and/or dioxygenases in the initial degradation of the aliphatic and aromatic fractions in PHCs.

INFECTIONS IN THE INTENSIVE CARE UNIT FOLLOWING LIVER TRANSPLANTATION: PROFILE OF A SINGLE CENTER

Introduction. Despite the advances in antibiotherapy and critical care management, infectious complications remain among the leading complications after liver transplantation related with mortality and morbidity. This study analysis the incidence and pattern of infections and possible prognostic factors of infectious compli- cations retrospectively in a single center. Patients and Methods. Results of 30 consecutive patients with a primary liver transplantation history in a single center between August 2011 and August 2012 and a positive culture result in the fi rst month in the ICU were analysed retrospectively. Results. During the fi rst 1 month stay in the ICU postoperatively 30 (13,63%) patients had at least 1 infection. Total number of infections were 68. Mortality rate of the infected patients was 53,3% (n = 16). Among these infections, 25 (36,76%) of them were in deep surgical sites. Eighteen of the 30 patients (60%) were infected with a single microorganism. Eleven patients (36,66%) had a single infection episode. Microorganism were gram negative in 52 (76,47%) of the infections, gram positive in 14 (20,58%) of the infections, rest of the 2 (2,94%) infections were due to Candidiasis. Among the possible risk factors contributing to mortality, there was a statistically signi fi cant dif- ference (p < 0,001) between the platelet counts of the mortality and surviving groups of the patients. Conclu- sion. Infections are among the preventable risk factors for mortality and morbidity after liver transplantation. Our data reveals a signi fi cant relation between trombocytopenia and mortality among the infected patients. Further studies focusing on this relation would expose the mechanisms and any possible contribution in cli- nical management of the patients.

A new chemoenzymatic approach to the synthesis of Latanoprost and Bimatoprost

Bimatoprost (1) and Latanoprost (2) are prostaglandin analogues widely used for glaucoma treatment. We have developed a new chemoenzymatic synthesis for 1 and 2, which utilizes a highly stereoselective sequence of biotransformations catalyzed by enzymes belonging to a single microorganism (the yeast Pichia anomala). The original synthesis, starting from (–)-Corey lactone benzoate (3aR,4R,5R,6aS)-3, was modified by replacing three synthetic steps (CC reduction, stereoselective CO reduction and hydrolysis/deprotection of the benzoate ester) with a one-pot, three-enzymes reaction. The overall biotransformation gave good yields and it was highly stereoselective; noteworthy, by engineering the reaction medium, CC reduction could be modulated so that unsaturated (3aR,4R,5R,6aS,3′S)-6 or saturated intermediate (3aR,4R,5R,6aS,3′R)-7 could be preferentially obtained.

Five-year assessment of causative agents and antibiotic resistances in urinary tract infections.

To show the distribution and changes of causative agents of urinary tract infections in children and resistance rates by years and select the most appropriate antibiotics. In this study, the Başkent University Alanya Research and Application Hospital automation system microbiology recording book was screened retrospectively. Growth of a single microorganism above 105 colonies (cfu/mL) was included in the assessment. Throughout the study, 10 691 urinary cultures were studies and growth was found in 392 (3.7%). Three hundred and nine (78.8%) of the samples with growth belonged to girls. Growth was found in the neonatal period in 32 patients (8.2%). The most commonly isolated microorganism was Escherichia coli (E. coli) which was found in 68.4% of the patients. Klebsiella spp. were found with a rate of 12.0%; Enterobacter spp. were found with a rate of 10.7% and proteus spp. were found with a rate of 5.1%. Resistance to cefalotin (62.1%), trimethoprim-sulfamethoxasole (43.1%), amoxycillin-clavulanate (34.8%), ampicillin (30.4%), cefixim (26.3%) and nitrofurantoin (3.6%) was found in E. coli species. The antibiotic which had the highest resistance rate was ampicillin with a rate of 93.2% for klebsiella and 83.4% for enterobacter. Klebsiella spp. were the most commonly grown pathogens in newborns (40.6%). In a follow-up period of 5 years, the resistance of E. coli to amoxycillin-clavulanate regressed from 40.3% to 31.3%, while the resistance to trimethoprim-sulfamethoxasole (TMP-SMX) regressed from 45.6% to 34.7%. A high resistance against first-generation cephalosporins, ampicillin, amoxycillin-clavulanate and TMP-SMX which are the first-line antibiotics in childhood urinary tract infections was found. Carbapenem (meropenem, imipenem) resistance was not found in our center. Nitrofurantoin, aminoglycosides and cefixime can be recommended for empirical treatment in our hospital because of low resistance. Antibiotic treatment should be redecided according to in vitro antibiotic sensitivity results.

Spatial and temporal distribution and characteristics of eukaryote-dominated microbial biofilms in an acid mine drainage environment: implications for development of iron-rich stromatolites

Spatial and temporal distribution and characteristics of three eukaryotic biofilms were monitored for an 18-month period in an acid mine drainage environment at the Green Valley coalmine in Indiana, USA. Each biofilm is dominated (>90 %) by a single eukaryotic microorganism based on enumeration: Euglena mutabilis, the diatom species Nitzschia tubicola, and a filamentous alga belonging to the genus Klebsormidium sp. The E. mutabilis-dominated biofilm occurs year round, covering up to 100 % of the channel bottom in spring and fall. The N. tubicola-dominated biofilm is less abundant, exists as small patches in spring and fall, expands from these patches to cover up to 50 % of the channel bottom in June, and is absent in winter. The Klebsormidium-dominated biofilm is restricted to small patches covering <5 % of the channel bottom from spring through fall and is absent in winter. Also present are floating microbial scum layers. The eukaryotic biofilms and scum layers contribute to the attenuation of precipitates and to the formation of organosedimentary structures, or stromatolites, by trapping and binding chemical precipitates via aerotaxis and phototaxis and by serving as a medium for passive accumulation of precipitates. Each stromatolite layer represents the morphological characteristics of each eukaryotic biofilm that served as the architect of the layer and the time of year the biofilm populated the channel. Processes involved in stromatolite formation also attenuate chemical sediments by binding them to the channel bottom and prior stromatolite surface rather than allowing them to be carried to the adjacent drainage system where they may become bioavailable to other forms of life.

Invasion of endothelial cells and arthritogenic potential of endocarditis-associated Corynebacterium diphtheriae

Although infection by Corynebacterium diphtheriae is a model of extracellular mucosal pathogenesis, different clones have been also associated with invasive infections such as sepsis, endocarditis, septic arthritis and osteomyelitis. The mechanisms that promote C. diphtheriae infection and haematogenic dissemination need further investigation. In this study we evaluated the association and invasion mechanisms with human umbilical vein endothelial cells (HUVECs) and experimental arthritis in mice of endocarditis-associated strains and control non-invasive strains. C. diphtheriae strains were able to adhere to and invade HUVECs at different levels. The endocarditis-associated strains displayed an aggregative adherence pattern and a higher number of internalized viable cells in HUVECs. Transmission electron microscopy (TEM) analysis revealed intracellular bacteria free in the cytoplasm and/or contained in a host-membrane-confined compartment as single micro-organisms. Data showed bacterial internalization dependent on microfilament and microtubule stability and involvement of protein phosphorylation in the HUVEC signalling pathway. A high number of affected joints and high arthritis index in addition to the histopathological features indicated a strain-dependent ability of C. diphtheriae to cause severe polyarthritis. A correlation between the arthritis index and increased systemic levels of IL-6 and TNF-α was observed for endocarditis-associated strains. In conclusion, higher incidence of potential mechanisms by which C. diphtheriae may access the bloodstream through the endothelial barrier and stimulate the production of pro-inflammatory cytokines such as IL-6 and TNF-α, in addition to the ability to affect the joints and induce arthritis through haematogenic spread are thought to be related to the pathogenesis of endocarditis-associated strains.

Variance decomposition: A tool enabling strategic improvement of the precision of analytical recovery and concentration estimates associated with microorganism enumeration methods

Concentrations of particular types of microorganisms are commonly measured in various waters, yet the accuracy and precision of reported microorganism concentration values are often questioned due to the imperfect analytical recovery of quantitative microbiological methods and the considerable variation among fully replicated measurements. The random error in analytical recovery estimates and unbiased concentration estimates may be attributable to several sources, and knowing the relative contribution from each source can facilitate strategic design of experiments to yield more precise data or provide an acceptable level of information with fewer data. Herein, variance decomposition using the law of total variance is applied to previously published probabilistic models to explore the relative contributions of various sources of random error and to develop tools to aid experimental design. This work focuses upon enumeration-based methods with imperfect analytical recovery (such as enumeration of Cryptosporidium oocysts), but the results also yield insights about plating methods and microbial methods in general. Using two hypothetical analytical recovery profiles, the variance decomposition method is used to explore 1) the design of an experiment to quantify variation in analytical recovery (including the size and precision of seeding suspensions and the number of samples), and 2) the design of an experiment to estimate a single microorganism concentration (including sample volume, effects of improving analytical recovery, and replication). In one illustrative example, a strategically designed analytical recovery experiment with 6 seeded samples would provide as much information as an alternative experiment with 15 seeded samples. Several examples of diminishing returns are illustrated to show that efforts to reduce error in analytical recovery and concentration estimates can have negligible effect if they are directed at trivial error sources.