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Live vaccines have long been known to trigger far more vigorous immune responses than their killed counterparts. This has been attributed to the ability of live microorganisms to replicate and express specialized virulence factors that facilitate invasion and infection of their hosts. However, protective immunization can often be achieved with a single injection of live, but not dead, attenuated microorganisms stripped of their virulence factors. Pathogen-associated molecular patterns (PAMPs), which are detected by the immune system, are present in both live and killed vaccines, indicating that certain poorly characterized aspects of live microorganisms, not incorporated in dead vaccines, are particularly effective at inducing protective immunity. Here we show that the mammalian innate immune system can directly sense microbial viability through detection of a special class of viability-associated PAMPs (vita-PAMPs). We identify prokaryotic messenger RNA as a vita-PAMP present only in viable bacteria, the recognition of which elicits a unique innate response and a robust adaptive antibody response. Notably, the innate response evoked by viability and prokaryotic mRNA was thus far considered to be reserved for pathogenic bacteria, but we show that even non-pathogenic bacteria in sterile tissues can trigger similar responses, provided that they are alive. Thus, the immune system actively gauges the infectious risk by searching PAMPs for signatures of microbial life and thus infectivity. Detection of vita-PAMPs triggers a state of alert not warranted for dead bacteria. Vaccine formulations that incorporate vita-PAMPs could thus combine the superior protection of live vaccines with the safety of dead vaccines.
High-throughput sequencing provides a powerful window into the structural and functional profiling of microbial communities, but it is unable to characterize only the viable portion of microbial communities at scale. There is as yet not one best solution to this problem. Previous studies have established viability assessments using propidium monoazide (PMA) treatment coupled with downstream molecular profiling (e.g., qPCR or sequencing). While these studies have met with moderate success, most of them focused on the resulting "viable" communities without systematic evaluations of the technique. Here, we present our work to rigorously benchmark "PMA-seq" (PMA treatment followed by 16S rRNA gene amplicon sequencing) for viability assessment in synthetic and realistic microbial communities.
Constitutive cell-autonomous immunity in metazoans predates interferon-inducible immunity and comprises primordial innate defense. Phagocytes mobilize interferon-inducible responses upon engagement of well-characterized signaling pathways by pathogen-associated molecular patterns (PAMPs). The signals controlling deployment of constitutive cell-autonomous responses during infection have remained elusive. Vita-PAMPs denote microbial viability, signaling the danger of cellular exploitation by intracellular pathogens. We show that cyclic-di-adenosine monophosphate in live Gram-positive bacteria is a vita-PAMP, engaging the innate sensor stimulator of interferon genes (STING) to mediate endoplasmic reticulum (ER) stress. Subsequent inactivation of the mechanistic target of rapamycin mobilizes autophagy, which sequesters stressed ER membranes, resolves ER stress, and curtails phagocyte death. This vita-PAMP-induced ER-phagy additionally orchestrates an interferon response by localizing ER-resident STING to autophagosomes. Our findings identify stress-mediated ER-phagy as a cell-autonomous response mobilized by STING-dependent sensing of a specific vita-PAMP and elucidate how innate receptors engage multilayered homeostatic mechanisms to promote immunity and survival after infection.
Today, probiotics are predominantly used in liquid or semi-solid functionalized foods, showing a rapid loss of cell viability. Due to the increasing spread of antibiotic resistance, probiotics are promising in pharmaceutical development because of their antimicrobial effects. This increases the formulation requirements, e.g., the need for an enhanced shelf life that is achieved by drying, mainly by lyophilization. For oral administration, the process chain for production of tablets containing microorganisms is of high interest and, thus, was investigated in this study. Lyophilization as an initial process step showed low cell survival of only 12.8%. However, the addition of cryoprotectants enabled survival rates up to 42.9%. Subsequently, the dried cells were gently milled. This powder was tableted directly or after mixing with excipients microcrystalline cellulose, dicalcium phosphate or lactose. Survival rates during tableting varied between 1.4% and 24.1%, depending on the formulation and the applied compaction stress. More detailed analysis of the tablet properties showed advantages of excipients in respect of cell survival and tablet mechanical strength. Maximum overall survival rate along the complete manufacturing process was >5%, enabling doses of 6 × 108 colony forming units per gram (CFU gtotal-1), including cryoprotectants and excipients.
Live vaccines historically afford superior protection, yet the cellular and molecular mechanisms mediating protective immunity remain unclear. Here we found that vaccination of mice with live, but not dead, Gram-negative bacteria heightened follicular T helper cell (Tfh) differentiation, germinal center formation, and protective antibody production through the signaling adaptor TRIF. Complementing the dead vaccine with an innate signature of bacterial viability, bacterial RNA, recapitulated these responses. The interferon (IFN) and inflammasome pathways downstream of TRIF orchestrated Tfh responses extrinsically to B cells and classical dendritic cells. Instead, CX3CR1+CCR2- monocytes instructed Tfh differentiation through interleukin-1β (IL-1β), a tightly regulated cytokine secreted upon TRIF-dependent IFN licensing of the inflammasome. Hierarchical production of IFN-β and IL-1β dictated Tfh differentiation and elicited the augmented humoral responses characteristic of live vaccines. These findings identify bacterial RNA, an innate signature of microbial viability, as a trigger for Tfh differentiation and suggest new approaches toward vaccine formulations for coordinating augmented Tfh and B cell responses.
Tetracycline (TC) is a broad-spectrum antibiotic compound. Wastewater with TC may have an adverse effect on ecosystems. Riboflavin-5'-phosphate (FMN or flavin mononucleotide) is a non-toxic product of the phosphorylation of vitamin B2 and is required for the proper functioning of the humans. FMN is sensitized to ultraviolet (UV) and blue light radiation, as evidenced by the generation of reactive oxygen species (ROS). This study inspects feasible applications of blue light on FMN so as to develop a valid way of degrading TC by FMN photolysis. We used the increased rate of bacterial survival as a practical indicator of antibiotic degradation. TC in the presence of FMN solution decomposed completely after 20 W/m2 of blue light irradiation (TCF treatment), and the degradation of TC (D-TCF) occurred after the photolytic process. After TCF treatment, colony-forming units (CFUs) of Escherichia coli (E. coli) were determined for the D-TCF solution. The CFU of E. coli preservation was 93.2% of the D-TCF solution (50 μg/mL of TC in the presence of 114 μg/mL of FMN solution treated with 20 W/m2 of blue light irradiation at 25 °C for 1 h) cultivation. The mass spectrum of D-TCF showed diagnostic ion signals at m/z 431.0 and 414.0 Da. The molecular formula of D-TCF was C21H22N2O8, and the exact mass was 430.44 g/mol. TC degradation by FMN photolysis can significantly decrease the antimicrobial ability of TC. The results expressed here regarding the influence of FMN photolysis on TC degradation offer an environmentally sound wastewater treatment method.
The erbium-doped yttrium aluminum garnet (Er:YAG) laser is used to treat periodontal disease; however, its effectiveness at killing oral bacteria is not well known. Furthermore, the compounding effect of the combination of a laser treatment and irrigation methods with antimicrobials on bacterial viability is yet to be determined. The purpose of this in vitro study was to evaluate the effect of the Er:YAG laser with irrigation using chlorhexidine (CHX), hydrogen peroxide (H2O2), or sodium hypochlorite (NaOCl) on the viability of oral bacteria. Three bacterial species were used in our study: Streptococcus gordonii, Fusobacterium nucleatum, and Porphyromonas gingivalis. Bacteria were grown in an anaerobic chamber in brain heart infusion broth and incubated at 37 °C. Bacterial samples with an OD of 0.5 were irradiated with the Er:YAG laser at 2940 nm using a 400-µm Varian tip. The experiment was repeated four times using these parameters: 40 mJ, 40 Hz, and 1.6 W for 20 seconds with the 300 µs short pulse duration in contact mode. Treatment groups consisted of the following: (1) no treatment, (2) 0.5% H2O2 alone, (3) 0.5% NaOCl alone, (4) 0.03% CHX alone, (5) Er:YAG irradiation alone, (6) Er:YAG irradiation with 0.5% H2O2, (7) Er:YAG irradiation with 0.5% NaOCl, and (8) Er:YAG irradiation with 0.03% CHX. Microbial viability was determined through plating and colony counts and calculated into CFU/ml. Statistical analysis was done using a two-tailed paired t-test. The use of the Er:YAG laser alone failed to show statistically significant antibacterial activity against any of bacteria. The most effective mono-treatment with irrigation solutions for all three bacteria were 0.5% H2O2 and 0.5% NaOCl (p < 0.001 for each solution). Irrigation with 0.03% CHX was most effective against F. nucleatum (p < 0.01) and less against P. gingivalis and S. gordonii and showed the least antibacterial action alone but improved significantly in combination therapy (p < 0.05). The combined treatment with the Er:YAG showed the greatest and most significant improvement in the reduction of bacterial viability compared to any other treatment group (p < 0.05 for each combined treatment). Irradiation with the Er:YAG laser with the addition of 0.5% H2O2, 0.5% NaOCl, or 0.03% CHX under a short working time (20 s) resulted in a significant reduction of bacterial viability for all three bacterial species compared with any single treatment option. The combination of irradiation with the Er:YAG laser with the addition of 0.5% H2O2, 0.5% NaOCl, or 0.03% CHX resulted in a larger reduction of bacterial survival when compared to monotherapies with antimicrobial solutions or laser. The combination of the Er:YAG laser with a low concentration irrigant solution of 0.5% H2O2, 0.5% NaOCl, or 0.03% CHX could be an effective treatment protocol for the reduction of periodontal pathogens and thus suitable treatment for non-surgical periodontal therapy.
The composite hydrogels were produced using the solution casting method due to the non-toxic and biocompatible nature of chitosan (CS)/polyvinyl alcohol (PVA). The best composition was chosen and crosslinked with tetraethyl orthosilicate (TEOS), after which different amounts of graphene oxide (GO) were added to develop composite hydrogels. Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), atomic force microscopy (AFM) and contact angle was used to analyze the hydrogels. The samples were also evaluated for swelling abilities in various mediums. The drug release profile was studied in phosphate-buffered saline (PBS) at a pH of 7.4. To predict the mechanism of drug release, the data were fitted into kinetic models. Finally, antibacterial activity and cell viability data were obtained. FTIR studies revealed the successful synthesis of CS/PVA hydrogels and GO/CS/PVA in hydrogel composite. SEM showed no phase separation of the polymers, whereas AFM showed a decrease in surface roughness with an increase in GO content. 100 µL of crosslinker was the critical concentration at which the sample displayed excellent swelling and preserved its structure. Both the crosslinked and composite hydrogel showed good swelling. The most acceptable mechanism of drug release is diffusion-controlled, and it obeys Fick's law of diffusion for drug released. The best fitting of the zero-order, Hixson-Crowell and Higuchi models supported our assumption. The GO/CS/PVA hydrogel composite showed better antibacterial and cell viability behaviors. They can be better biomaterials in biomedical applications.
The therapeutic potential of faecal microbiota transplantation (FMT) is under investigation for a range of inflammatory conditions. While mechanisms of benefit are poorly understood, most models rely on the viability of transplanted microbes. We hypothesised that protocols commonly used in the preparation of faecal transplants will substantially reduce the number, diversity and functional potential of viable microbes.
Recent evidences highlighted the presence of Lactococcus lactis during late cheese ripening. For this reason, the role of this microorganism, well known as dairy starter, should be reconsidered throughout cheese manufacturing and ripening. Thus, the main objective of this study was to develop a RT-qPCR protocol for the detection, quantification and determination of the viability of L. lactis in ripened cheese samples by direct analysis of microbial nucleic acids. Standard curves were constructed for the specific quantification of L. lactis in cheese matrices and good results in terms of selectivity, correlation coefficient and efficiency were obtained. Thirty-three ripened cheeses were analyzed and, on the basis of RNA analysis, twelve samples showed 106 to 108 CFU of L. lactis per gram of product, thirteen from 103 to 105 CFU/g, and in eight cheeses, L. lactis was not detected. Traditional plating on M17 medium led to loads ranging from 105 to 109 CFU/g, including the cheese samples where no L. lactis was found by RT-qPCR. From these cheeses, none of the colonies isolated on M17 medium was identified as L. lactis species. These data could be interpreted as a lack of selectivity of M17 medium where colony growth is not always related to lactococcal species. At the same time, the absence or low abundance of L. lactis isolates on M17 medium from cheese where L. lactis was detected by RT-qPCR support the hypothesis that L. lactis starter populations are mainly present in viable but not culturable state during ripening and, for this reason, culture-dependent methods have to be supplemented with direct analysis of cheese.
Autophagy is an essential cellular mechanism that degrades cytoplasmic proteins and organelles to recycle their components. Moreover, autophagy is essential for preimplantation development in mammals. Here we show that autophagy is also important for reprogramming in somatic cell nuclear transfer (SCNT). Our data indicate that unlike fertilized oocytes, autophagy is not triggered in SCNT embryos during 6 hours of activation. Mechanistically, the inhibited autophagic induction during SCNT activation is due to the cytochalasin B (CB) caused depolymerization of actin filaments. In this study, we induced autophagy during SCNT activation by rapamycin and pp242, which could restore the expected level of autophagy and significantly enhance the development of SCNT embryos to the blastocyst stage when compared with the control (68.5% and 68.7% vs. 41.5%, P < 0.05). Furthermore, the treatment of rapamycin and pp242 accelerates active DNA demethylation indicated by the conversion of 5 mC to 5 hmC, and treatment of rapamycin improves degradation of maternal mRNA as well. Thus, our findings reveal that autophagy is important for development of SCNT embryos and inhibited autophagic induction during SCNT activation might be one of the serious causes of low efficiency of SCNT.
Sponges host complex microbial communities of recognized ecological and biotechnological importance. Extensive cultivation efforts have been made to isolate sponge bacteria, but most still elude cultivation. To identify the bottlenecks of sponge bacterial cultivation, we combined high-throughput 16S rRNA gene sequencing with a variety of cultivation media and incubation conditions. We aimed to determine the extent to which sample processing and cultivation conditions can impact bacterial viability and recovery in culture. We isolated 325 sponge bacteria from six specimens of Cymbastela concentrica and three specimens of Scopalina sp. These isolates were distributed over 37 different genera and 47 operational taxonomic units (defined at 97% 16S rRNA gene sequence identity). The cultivable bacterial community was highly specific to its sponge host and different media compositions yielded distinct microbial isolates. Around 97% of the isolates could be detected in the original sponge and represented a large but highly variable proportion (0.5-92% total abundance, depending on sponge species) of viable bacteria obtained after sample processing, as determined by propidium monoazide selective DNA modification of compromised cells. Our results show that the most abundant viable bacteria are also the most predominant groups found in cultivation, reflecting, to some extent, the relative abundances of the viable bacterial community, rather than the overall community estimated by direct molecular approaches. Cultivation is therefore shaped not only by the growth conditions provided, but also by the different cell viabilities of the bacteria that constitute the cultivation inoculum. These observations highlight the need to perform experiments to assess each method of sample processing for its accurate representation of the actual in situ bacterial community and its yield of viable cells.
Filamentous bacteria of the genus Streptomyces produce a large arsenal of industrially relevant antibiotics and enzymes. The industrial production of these molecules occurs in large fermenters, where many streptomycetes form dense mycelial networks called pellets. Pellets are characterized by slow growth and inefficient nutrient transfer and therefore regarded as undesirable from the perspective of productivity. Although non-pelleting strains have increased growth rates, their morphology also leads to a dramatic increase in the viscosity of the culture broth, which negatively impacts the process dynamics.
Bacterial vaginosis (BV) is the most common vaginal infection among women of reproductive age. A hallmark of BV is the presence of a highly structured polymicrobial biofilm on the vaginal epithelium, presumably initiated by facultative anaerobes of the genus Gardnerella, which then becomes a scaffold for other species to adhere to. One of the species often found incorporated in Gardnerella mediated biofilms is Atopobium vaginae. Interestingly, A. vaginae is very rarely found without the presence of Gardnerella. However, not much is known regarding the interactions between A. vaginae and Gardnerella species. This study assessed biological interactions between Gardnerella vaginalis and A. vaginae. In our in vitro model, by using specific Gardnerella and A. vaginae Peptide Nucleic Acid (PNA)-Fluorescence In Situ Hybridization (FISH) probes, we confirmed that A. vaginae was able to incorporate a pre-formed G. vaginalis biofilm, accounting for up to 20% of the total number of biofilm cells. However, our findings showed that almost 92% of A. vaginae cells lost viability after 48 h of mono-species planktonic growth, but were able to maintain viability when co-cultured with Gardnerella or after pre-conditioning with cell-free supernatant of Gardnerella cultures. While the in vitro conditions are very different from the in vivo microenvironment, this study contributes to a better understanding of why A. vaginae vaginal colonization rarely occurs in the absence of Gardnerella. Overall, this highlights the importance of microbial interactions between BV-associated bacteria and demands more studies focused on the polymicrobial bacterial communities found in BV.
In bacteriology, the ability to grow in selective media and to form colonies on nutrient agar plates is routinely used as a retrospective criterion for the detection of living bacteria. However, the utilization of indicators for bacterial viability-such as the presence of specific transcripts or membrane integrity-would overcome bias introduced by cultivation and reduces the time span of analysis from initiation to read out. Therefore, we investigated the correlation between transcriptional activity, membrane integrity and cultivation-based viability in the Gram-positive model bacterium Bacillus subtilis.
Nasopharyngeal swabs are considered the gold-standard sample type for the detection of Streptococcus pneumoniae carriage, but recent studies have demonstrated the utility of saliva in improving the detection of carriage in adults. Saliva is generally collected in its raw, unsupplemented state, unlike nasopharyngeal swabs, which are collected into stabilizing transport media. Few data exist regarding the stability of pneumococci in unsupplemented saliva during transport and laboratory storage. We therefore evaluated the effect of storage conditions on the detection of pneumococci in saliva samples using strains representing eight pneumococcal serotypes. The bacteria were spiked into raw saliva from asymptomatic individuals, and we assessed sample viability after storage at 4°C, room temperature, and 30°C for up to 72 h; at 40°C for 24 h; and following three freeze-thaw cycles. We observed little decrease in pneumococcal detection following culture enrichment and quantitative PCR (qPCR) detection of the piaB and lytA genes compared to testing fresh samples, indicating the prolonged viability of pneumococci in neat saliva samples. This sample stability makes saliva a viable sample type for pneumococcal carriage studies conducted in remote or low-resource settings and provides insight into the effect of the storage of saliva samples in the laboratory. IMPORTANCE For pneumococcal carriage studies, saliva is a sample type that can overcome some of the issues typically seen with nasopharyngeal and oropharyngeal swabs. Understanding the limitations of saliva as a sample type is important for maximizing its use. This study sought to better understand how different storage conditions and freeze-thaw cycles affect pneumococcal survival over time. These findings support the use of saliva as an alternative sample type for pneumococcal carriage studies, particularly in remote or low-resource settings with reduced access to health care facilities.
Neuropeptide hormone oxytocin has roles in social bonding, energy metabolism, and wound healing contributing to good physical, mental and social health. It was previously shown that feeding of a human commensal microbe Lactobacillus reuteri (L. reuteri) is sufficient to up-regulate endogenous oxytocin levels and improve wound healing capacity in mice. Here we show that oral L. reuteri-induced skin wound repair benefits extend to human subjects. Further, dietary supplementation with a sterile lysate of this microbe alone is sufficient to boost systemic oxytocin levels and improve wound repair capacity. Oxytocin-producing cells were found to be increased in the caudal paraventricular nucleus [PVN] of the hypothalamus after feeding of a sterile lysed preparation of L. reuteri, coincident with lowered blood levels of stress hormone corticosterone and more rapid epidermal closure, in mouse models. We conclude that microbe viability is not essential for regulating host oxytocin levels. The results suggest that a peptide or metabolite produced by bacteria may modulate host oxytocin secretion for potential public or personalized health goals.
The application of beneficial, plant-associated microorganisms is a sustainable approach to improving crop performance in agriculture. However, microbial inoculants are often susceptible to prolonged periods of storage and deleterious environmental factors, which negatively impact their viability and ultimately limit efficacy in the field. This particularly concerns non-sporulating bacteria. To overcome this challenge, the availability of protective formulations is crucial. Numerous parameters influence the viability of microbial cells, with drying procedures generally being among the most critical ones. Thus, technological advances to attenuate the desiccation stress imposed on living cells are key to successful formulation development. In this review, we discuss the core aspects important to consider when aiming at high cell viability of non-sporulating bacteria to be applied as microbial inoculants in agriculture. We elaborate the suitability of commonly applied drying methods (freeze-drying, vacuum-drying, spray-drying, fluidized bed-drying, air-drying) and potential measures to prevent cell damage from desiccation (externally applied protectants, stress pre-conditioning, triggering of exopolysaccharide secretion, 'helper' strains). Furthermore, we point out methods for assessing bacterial viability, such as colony counting, spectrophotometry, microcalorimetry, flow cytometry and viability qPCR. Choosing appropriate technologies for maintenance of cell viability and evaluation thereof will render formulation development more efficient. This in turn will aid in utilizing the vast potential of promising, plant beneficial bacteria as sustainable alternatives to standard agrochemicals.
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