Viable Organisms Research Articles

Filifactor alocis modulates human neutrophil antimicrobial functional responses.

Filifactor alocis is a newly appreciated pathogen in periodontal diseases. Neutrophils are the predominant innate immune cell in the gingival crevice. In this study, we examined modulation of human neutrophil antimicrobial functions by F.alocis. Both non-opsonised and serum-opsonised F.alocis were engulfed by neutrophils but were not efficiently eliminated. Challenge of neutrophils with either non-opsonised or serum-opsonised F.alocis induced a minimal intracellular as well as extracellular respiratory burst response compared to opsonised Staphylococcus aureus and fMLF, respectively. However, pretreatment or simultaneous challenge of neutrophils with F.alocis did not affect the subsequent oxidative response to a particulate stimulus, suggesting that the inability to trigger the respiratory response was only localised to F.alocis phagosomes. In addition, although neutrophils engulfed live or heat-killed F.alocis with the same efficiency, heat-killed F.alocis elicited a higher intracellular respiratory burst response compared to viable organisms, along with decreased surface expression of CD35, a marker of secretory vesicles. F.alocis phagosomes remained immature by delayed and reduced recruitment of specific and azurophil granules, respectively. These results suggest that F.alocis withstands neutrophil antimicrobial responses by preventing intracellular ROS production, along with specific and azurophil granule recruitment to the bacterial phagosome.

Evaluating the Sporicidal Activity of Disinfectants against Clostridium difficile and Bacillus amyloliquefaciens Spores by Using the Improved Methods Based on ASTM E2197-11.

Spore-forming pathogenic bacteria, such as Clostridium difficile, are associated with nosocomial infection, leading to the increased use of sporicidal disinfectants, which impacts socioeconomic costs. However, C. difficile can be prevented using microorganisms such as Bacillus amyloliquefaciens, a prophylactic agent that has been proven to be effective against it in recent tests or it can be controlled by sporicidal disinfectants. These disinfectants against spores should be evaluated according to a known and recommended standard. Unfortunately, some newly manufactured disinfectants like Bioxy products have not yet been tested. ASTM E2197-11 is a standard test that uses stainless steel disks (1 cm in diameter) as carriers, and the performance of the test formulation is calculated by comparing the number of viable test organisms to that on the control carriers. Surface tests are preferable for evaluating disinfectants with sporicidal effects on hard surfaces. This study applies improved methods, based on the ASTM E2197-11 standard, for evaluating and comparing the sporicidal efficacies of several disinfectants against spores of C. difficile and B. amyloliquefaciens, which are used as the test organisms. With the improved method, all spores were recovered through vortexing and membrane filtration. The results show that chlorine-based products are effective in 5 min and Bioxy products at 5% w/v are effective in 10 min. Although Bioxy products may take longer to prove their effectiveness, their non-harmful effects to hospital surfaces and people have been well established in the literature.

MALDI-TOF mass spectrometry and high-consequence bacteria: safety and stability of biothreat bacterial sample testing in clinical diagnostic laboratories.

We considered the application of MALDI-TOF mass spectrometry for BSL-3 bacterial diagnostics, with a focus on the biosafety of live-culture direct-colony testing and the stability of stored extracts. Biosafety level 2 (BSL-2) bacterial species were used as surrogates for BSL-3 high-consequence pathogens in all live-culture MALDI-TOF experiments. Viable BSL-2 bacteria were isolated from MALDI-TOF mass spectrometry target plates after 'direct-colony' and 'on-plate' extraction testing, suggesting that the matrix chemicals alone cannot be considered sufficient to inactivate bacterial culture and spores in all samples. Sampling of the instrument interior after direct-colony analysis did not recover viable organisms, suggesting that any potential risks to the laboratory technician are associated with preparation of the MALDI-TOF target plate before or after testing. Secondly, a long-term stability study (3 years) of stored MALDI-TOF extracts showed that match scores can decrease below the threshold for reliable species identification (<1.7), which has implications for proficiency test panel item storage and distribution.

Loss of the deubiquitinase USP36 destabilizes the RNA helicase DHX33 and causes preimplantation lethality in mice

Deubiquitinases are proteases with a wide functional diversity that profoundly impact multiple biological processes. Among them, the ubiquitin-specific protease 36 (USP36) has been implicated in the regulation of nucleolar activity. However, its functional relevance in vivo has not yet been fully described. Here, we report the generation of an Usp36-deficient mouse model to examine the function of this enzyme. We show that Usp36 depletion is lethal in preimplantation mouse embryos, where it blocks the transition from morula to blastocyst during embryonic development. USP36 reduces the ubiquitination levels and increases the stability of the DEAH-box RNA helicase DHX33, which is critically involved in ribosomal RNA synthesis and mRNA translation. In agreement with this finding, O-propargyl-puromycin incorporation experiments, Northern blot, and electron microscopy analyses demonstrated the role of USP36 in ribosomal RNA and protein synthesis. Finally, we show that USP36 down-regulation alters cell proliferation in human cancer cells by inducing both apoptosis and cell cycle arrest, and that reducing DHX33 levels through short hairpin RNA interference has the same effect. Collectively, these results support that Usp36 is essential for cell and organism viability because of its role in ribosomal RNA processing and protein synthesis, which is mediated, at least in part, by regulating DHX33 stability.

Viable Pathogenic Organism Transportation and Recovery from a Low-Cost Support

There are two leading causes for rapid detection of pathogens: pathogenic transport and sample preparation. A variety of regulations is in place for the safe transport of pathogens from within the United States and internationally. Unfortunately, these methods require large amounts of paperwork, specialized shipping and handling practices, and costly materials. A new way to transport and store pathogens is by 903TM Protein Saver Cards. These cards allow for 5 samples, up to 100 μL per spot, to be transported safely due to the cards being dry and the pathogen still being viable for detection methods such as Polymerase Chain Reaction (PCR). In this presentation, we demonstrate the compatibility of these cards to maintain the genetic material of Listeria monocytogenes and Vibrio cholerae for lysing and detection applications. A spot of the protein card is cut, the bacteria is rehydrated, and then extracted from the card for cellular lysing using the Lyse-It® technology. Following cellular lysing, genomic DNA is detected using PCR. The positive results indicate that genetic material contained in the cell is still viable for specific identification of these pathogens. Within microwave cellular lysis and literature today remains the question of how the DNA is being fragmented. Literature suggests that reactive oxygen species, such as singlet oxygen, play a vital role in DNA fragmentation. We present evidence of increased and/or rapid microwave-induced singlet oxygen generation. Singlet oxygen is detected using Singlet Oxygen Sensor Green, where the endoperoxide increases the fluorescent intensity when excited by a 473 nm laser. This detection of singlet oxygen is indicative of its formation under microwave irradiation and therefore one player in the role of DNA fragmentation.

A Flow Cytometry Method for Assessing M. tuberculosis Responses to Antibiotics.

Traditional drug susceptibility methods can take several days or weeks of incubation between drug exposure and confirmation of sensitivity or resistance. In addition, these methods do not capture information about viable organisms that are not immediately culturable after drug exposure. Here, we present a rapid fluorescence detection method for assessing the susceptibility of M. tuberculosis to antibiotics. Fluorescent markers Calcein violet-AM and SYTOX-green are used for measuring cell viability or cell death and to capture information about the susceptibility of the whole population and not just those bacteria that can grow in media postexposure. Postexposure to the antibiotic, the method gives a rapid readout of drug susceptibility, as well as insights into the concentration and time-dependent drug activity following antibiotic exposure.

Update: Vaccines in primary immunodeficiency

Vaccines were originally developed to prevent or ameliorate infectious disease. As knowledge of immune function and appreciation of immunodeficiency has developed, researchers have used vaccine responses as a tool to characterize the phenotypes of patients exhibiting various syndromes. Thus it has become possible for a clinician to evaluate individual responses to vaccines to interrogate the immunocompetence of their patients. Although there have been many advances in these areas, we still have much to learn about the quantity and quality of humoral and cellular vaccine responses in healthy and immunodeficient subjects and how that knowledge can then be extrapolated to diagnostic purposes. Adverse effects of vaccines have been recognized for many years, especially the occurrence of infections caused by viable vaccine organisms in immunodeficient hosts. Nevertheless, vaccines are essential for disease prevention in immunodeficient patients, just as they are for healthy subjects. Clinicians must understand the appropriate and safe use of vaccines in patients with immunodeficiency. This review highlights some recent advances and ongoing challenges in application of vaccines for the diagnosis and treatment of immunodeficiencies.

Unraveling co-translational protein folding: Concepts and methods.

Advances in techniques such as nuclear magnetic resonance spectroscopy, cryo-electron microscopy, and single-molecule and time-resolved fluorescent approaches are transforming our ability to study co-translational protein folding both in vivo in living cells and in vitro in reconstituted cell-free translation systems. These approaches provide comprehensive information on the spatial organization and dynamics of nascent polypeptide chains and the kinetics of co-translational protein folding. This information has led to an improved understanding of the process of protein folding in living cells and should allow remaining key questions in the field, such as what structures are formed within nascent chains during protein synthesis and when, to be answered. Ultimately, studies using these techniques will facilitate development of a unified concept of protein folding, a process that is essential for proper cell function and organism viability. This review describes current methods for analysis of co-translational protein folding with an emphasis on some of the recently developed techniques that allow monitoring of co-translational protein folding in real-time.

Análisis de técnicas de recuento de Microorganismos.

El aislamiento y obtención de cultivos puros posiblemente hayan sido las bases más importantes en el desarrollo de la Microbiología. Los primeros trabajos en Microbiología se hicieron en unas condiciones experimentales que no permitían tener la certeza de que se hubieran obtenido cultivos puros. Más adelante, los microbiólogos observaron que, aparentemente, los microorganismos tenían una gran capacidad de variación en cuanto a su aspecto morfológico y características fisiológicas. Un cultivo puro es aquel que contiene una sola clase de microorganismo. En la práctica los cultivos puros son útiles por diferentes razones: mantienen los organismos viables, permiten hacer subcultivos para someterlos a diferentes análisis. Para obtener lo anterior es necesario recurrir a las técnicas de recuento. Existen métodos que permiten establecer el número de microorganismos en una muestra dada. Hay métodos que cuantifican el número de células, y existen otros que cuantifican la masa total de la población. El objetivo de este estudio es conocer las diversas técnicas utilizadas para el recuento de células de microorganismos, al igual que evaluar la efectividad de cada uno de estos métodos de conteo de células.

Efficacy of a Blend of Sulfuric Acid and Sodium Sulfate against Shiga Toxin–Producing Escherichia coli, Salmonella, and Nonpathogenic Escherichia coli Biotype I on Inoculated Prerigor Beef Surface Tissue

A study was conducted to investigate the efficacy of a sulfuric acid-sodium sulfate blend (SSS) against Escherichia coli O157:H7, non-O157 Shiga toxin-producing E. coli (STEC), Salmonella, and nonpathogenic E. coli biotype I on prerigor beef surface tissue. The suitability of using the nonpathogenic E. coli as a surrogate for in-plant validation studies was also determined by comparing the data obtained for the nonpathogenic inoculum with those for the pathogenic inocula. Prerigor beef tissue samples (10 by 10 cm) were inoculated (ca. 6 log CFU/cm2) on the adipose side in a laboratory-scale spray cabinet with multistrain mixtures of E. coli O157:H7 (5 strains), non-O157 STEC (12 strains), Salmonella (6 strains), or E. coli biotype I (5 strains). Treatment parameters evaluated were two SSS pH values (1.5 and 1.0) and two spray application pressures (13 and 22 lb/in2). Untreated inoculated beef tissue samples served as controls for initial bacterial populations. Overall, the SSS treatments lowered inoculated (6.1 to 6.4 log CFU/cm2) bacterial populations by 0.6 to 1.5 log CFU/cm2 (P < 0.05), depending on inoculum type and recovery medium. There were no main effects (P ≥ 0.05) of solution pH or spray application pressure when SSS was applied to samples inoculated with any of the tested E. coli inocula; however, solution pH did have a significant effect (P < 0.05) when SSS was applied to samples inoculated with Salmonella. Results indicated that the response of the nonpathogenic E. coli inoculum to the SSS treatments was similar (P ≥ 0.05) to that of the pathogenic inocula tested, making the E. coli biotype I strains viable surrogate organisms for in-plant validation of SSS efficacy on beef. The application of SSS at the tested parameters to prerigor beef surface tissue may be an effective intervention for controlling pathogens in a commercial beef harvest process.

Cooperation and cheating as innovation: insights from cellular societies.

The capacity to innovate is often considered a defining feature of human societies, but it is not a capacity that is unique to human societies: innovation occurs in cellular societies as well. Cellular societies such as multicellular bodies and microbial communities, including the human microbiome, are capable of innovation in response to novel opportunities and threats. Multicellularity represents a suite of innovations for cellular cooperation, but multicellularity also opened up novel opportunities for cells to cheat, exploiting the infrastructure and resources of the body. Multicellular bodies evolve less quickly than the cells within them, leaving them vulnerable to cellular innovations that can lead to cancer and infections. In order to counter these threats, multicellular bodies deploy additional innovations including the adaptive immune system and the development of partnerships with preferred microbial partners. What can we learn from examining these innovations in cooperation and cheating in cellular societies? First, innovation in social systems involves a constant tension between novel mechanisms that enable greater size and complexity of cooperative entities and novel ways of cheating. Second, cultivating cooperation with partners who can rapidly and effectively innovate (such as microbes) is important for large entities including multicellular bodies. And third, multicellularity enabled cells to manage risk socially, allowing organisms to survive in challenging environments where life would otherwise be impossible. Throughout, we ask how insights from cellular societies might be translated into new innovations in human health and medicine, promoting and protecting the cellular cooperation that makes us viable multicellular organisms.This article is part of the themed issue 'Process and pattern in innovations from cells to societies'.

Caprylic and Polygalacturonic Acid Combinations for Eradication of Microbial Organisms Embedded in Biofilm.

There is a need for non-antibiotic, antimicrobial compositions with low toxicity capable of broad-spectrum eradication of pathogenic biofilms in food preparation and healthcare settings. In this study we demonstrated complete biofilm eradication within 60 min with synergistic combinations of caprylic and polygalacturonic (PG) acids in an in vitro biofilm eradication model against representative hospital and foodborne infectious pathogen biofilms (methicillin-resistant Staphylococcus aureus, multidrug-resistant Pseudomonas aeruginosa, Candida albicans, Escherichia coli, and Salmonella enteritidis). Antimicrobial synergy against biofilms was demonstrated by quantifying viable organisms remaining in biofilms exposed to caprylic acid alone, PG acid alone, or combinations of the two. The combinations also synergistically inhibited growth of planktonic organisms. Toxicity of the combination was assessed in vitro on L929 fibroblasts incubated with extracts of caprylic and PG acid combinations using the Alamar Blue metabolic activity assay and the Trypan Blue exclusion cell viability assay. The extracts did not produce cytotoxic responses relative to untreated control fibroblasts.

The dissolvable bead: A novel in vitro biofilm model for evaluating antimicrobial resistance

In vitro biofilm assays are a vital first step in the assessment of therapeutic effectiveness. Current biofilm models have been found to be limited by throughput, reproducibility, and cost. We present a novel in vitro biofilm model, utilising a sodium alginate substratum for surface biofilm colony formation, which can be readily dissolved for accurate evaluation of viable organisms. The dissolving bead biofilm assay was evaluated using a range of clinically relevant strains. The reproducibility and responsiveness of the assay to an antimicrobial challenge was assessed using standardised methods. Cryo-scanning electron microscopy was used to image biofilm colonies. Biofilms were grown for 20h prior to testing. The model provides a reproducible and responsive assay to clinically-relevant antimicrobial challenges, as defined by established guidelines. Moreover cryo-scanning electron microscopy demonstrates that biofilm formation is localised exclusively to the alginate bead surface.Our results suggest that this simple model provides a robust and adaptable assay for the investigation of bacterial biofilms.

Biotesting of plasma-chemically activated water with the use of hydrobionts

The study has determined the influence of plasma-chemically activated water and its level of toxicity on test objects. It has been established that the reaction of biological objects is positive, which is reflected in their livelihood. The obtained research results show that the nature of the influence of water of different structures on the species composition of the selected test objects depends on the individual properties of each of them. The influence of plasma-chemically activated water on the functional properties of Riccia fluitans, L e mna m i nor L., Paramecium с audatum, Artemia salina and Cyprinus carpio has been investigated. The completed tests have helped supplement and substantiate the already existing idea about the properties of the plasma-chemically activated water and the possibilities of its use in the processing of raw materials and the manufacturing of finished products. In the course of the research, it was found that the Paramecium с audatum survival was 87–89.5 %, with no toxic effects of the plasma-chemically activated water on these objects. The cultivation of Artemia salina in water that had been pre-treated by the proposed method showed that the number of viable organisms had increased by 60–70 % compared with the reference. The obtained results of studying the influence of the plasma-chemically activated water on the development of Riccia fluitans and L e mna m i nor L. indicate that the activation of growth processes is 7–39 % higher than in the reference. It has been determined that the cultivation of Cyprinus carpio in the treated water contributes to increasing the survival rate as it improved the test objects by 13 % and 6 %, respectively, as to the reference. Using the method of gas-discharge diagnostics has shown that the treatment of water with nonthermal plasma for 30 minutes contributes to increasing its biopotential. Consequently, the obtained results indicate the expediency of using plasma-chemically activated water for the production of food and feed. The established positive changes support information on the safety of using plasma-chemically activated water as a component of the food chain and prove the practical value of a comprehensive approach to innovative methods of preparing raw materials.

Hsp83 loss suppresses proteasomal activity resulting in an upregulation of caspase-dependent compensatory autophagy

ABSTRACTThe 2 main degradative pathways that contribute to proteostasis are the ubiquitin-proteasome system and autophagy but how they are molecularly coordinated is not well understood. Here, we demonstrate an essential role for an effector caspase in the activation of compensatory autophagy when proteasomal activity is compromised. Functional loss of Hsp83, the Drosophila ortholog of human HSP90 (heat shock protein 90), resulted in reduced proteasomal activity and elevated levels of the effector caspase Dcp-1. Surprisingly, genetic analyses showed that the caspase was not required for cell death in this context, but instead was essential for the ensuing compensatory autophagy, female fertility, and organism viability. The zymogen pro-Dcp-1 was found to interact with Hsp83 and undergo proteasomal regulation in an Hsp83-dependent manner. Our work not only reveals unappreciated roles for Hsp83 in proteasomal activity and regulation of Dcp-1, but identifies an effector caspase as a key regulatory factor for sustaining adaptation to cell stress in vivo.

A smart device application for the automated determination of E. coli colonies on agar plates.

The manual counting of colonies on agar plates to estimate the number of viable organisms (so-called colony-forming units-CFUs) in a defined sample is a commonly used method in microbiological laboratories. The automation of this arduous and time-consuming process through benchtop devices with integrated image processing capability addresses the need for faster and higher sample throughput and more accuracy. While benchtop colony counter solutions are often bulky and expensive, we investigated a cost-effective way to automate the colony counting process with smart devices using their inbuilt camera features and a server-based image processing algorithm. The performance of the developed solution is compared to a commercially available smartphone colony counter app and the manual counts of two scientists trained in biological experiments. The comparisons show a high accuracy of the presented system and demonstrate the potential of smart devices to displace well-established laboratory equipment.

Composting to sanitize plant‐based waste infected with organisms of plant health importance

The potential for using the composting process to sanitize plant waste infected with one of three plant pathogens was investigated using bench‐scale composting equipment. Two of these pathogens, the potato wart disease fungus Synchytrium endobioticum and Potato spindle tuber viroid (PSTVd) are currently subject to European quarantine regulations. The third, Polymyxa betae, a parasite of sugar beet, is regulated in some European countries when in association with Beet necrotic yellow vein virus (BNYVV), the causal organism of rhizomania disease of sugar beet. Survival of test organisms following various combinations of compost temperature, exposure time and moisture was determined using RNA‐based detection methodology and/or plant‐based bioassays. Mathematically definable relationships between compost treatment (temperature/time) and organism viability were identified for P. betae and S. endobioticum; these give some indication of the practicality of using composting for dealing with infected wastes. However, for PSTVd, the considerable variability in measured susceptibility of the viroid to the composting process meant that no such definable relationship could be determined and further work would be needed to extrapolate to practical situations.

Structural basis for dolichylphosphate mannose biosynthesis

Protein glycosylation is a critical protein modification. In biogenic membranes of eukaryotes and archaea, these reactions require activated mannose in the form of the lipid conjugate dolichylphosphate mannose (Dol-P-Man). The membrane protein dolichylphosphate mannose synthase (DPMS) catalyzes the reaction whereby mannose is transferred from GDP-mannose to the dolichol carrier Dol-P, to yield Dol-P-Man. Failure to produce or utilize Dol-P-Man compromises organism viability, and in humans, several mutations in the human dpm1 gene lead to congenital disorders of glycosylation (CDG). Here, we report three high-resolution crystal structures of archaeal DPMS from Pyrococcus furiosus, in complex with nucleotide, donor, and glycolipid product. The structures offer snapshots along the catalytic cycle, and reveal how lipid binding couples to movements of interface helices, metal binding, and acceptor loop dynamics to control critical events leading to Dol-P-Man synthesis. The structures also rationalize the loss of dolichylphosphate mannose synthase function in dpm1-associated CDG.

Pheomelanin molecular vibration is associated with mitochondrial ROS production in melanocytes and systemic oxidative stress and damage.

Vibrations in covalent bonds affect electron delocalization within molecules, as reported in polymers. If synthesized by living cells, the electron delocalization of polymers affects the stabilization of cellular free radicals, but biomolecular vibration has never been considered a potential source of cytotoxicity. Here we show that the vibrational characteristics of natural pheomelanin and eumelanin contribute to feather color expression in four poultry breeds with different melanin-based pigmentation patterns, but only the vibrational characteristics of pheomelanin are related to the production of reactive oxygen species (ROS) in the mitochondria of melanocytes and to systemic levels of cellular oxidative stress and damage. This association may be explained by the close physical contact existing between mitochondria and melanosomes, and reveals an unprecedented factor affecting the viability of organisms through their pigmentation. These findings open a new avenue for understanding the mechanism linking pheomelanin synthesis to human melanoma risk.

Calcium signaling-related genes in Penaeus monodon respond to abiotic stress and pathogenic bacteria common in aquaculture

Stress of cultured species is a common problem, affecting the host health, in aquatic farming. It occurs when interactions among the three elements – host, environment and pathogens, are not in balance, particularly under intensive culturing conditions (de la Vega et al., 2006). Hosts respond to stress by triggering cellular Ca2+ signaling to activate many downstream pathways for their functions in order to mitigate the stress effect. Calreticulin (CRT), Calnexin (CNX) and Endoplasmic reticulum protein 57 (ERp57) are involved in Ca2+ signaling in stress response mechanisms and are essential for survival and viability of organisms under stress conditions. In this study, the full length cDNA of CNX and ERp57 in the giant tiger shrimp (Penaeus monodon) were identified. Amino acid sequences deduced from both cDNA showed significantly high similarity with those present in other crustaceans. One and two forms of PmCNX and PmERp57 transcripts were found, respectively. For PmERp57, its genomic sequence was additionally identified. It spanned 8257 base pairs (bp), composing of 10 exons (84–226bp long) and 9 introns (93–2787bp long). Both genes were expressed in all examined tissues of both juvenile and broodstock P. monodon. Effects of Vibrio harveyi and abotic stress commonly found in aquaculture on expression of PmCRT, PmCNX and PmERp57 were examined in hemocytes by quantitative real-time PCR. High temperature, hypo- and hyper-osmosis and V. harveyi infection induced expression all of the three genes (P<0.05). In contrast, analysis of hypoxia effect on expression of the Ca2+ signaling-related genes, which was firstly studied in aquatic species, showed to suppress their expression (P<0.05). Results from this study suggested that PmCRT, PmCNX and PmERp57 are related to stress response mechanisms and immune system of P. monodon. Their expression is therefore a potential candidate for markers to detect or monitor the early sign of shrimp's stress and immunity changes in farming.