Live Bacteria Research Articles

Electrochromogenic Detection of Live Bacteria Using Soluble and Insoluble Prussian Blue.

Microbial detection is crucial for the control and prevention of infectious diseases, being one of the leading causes of mortality worldwide. Among the techniques developed for bacterial detection, those based on metabolic indicators are progressively gaining interest due to their simplicity, adaptability, and, most importantly, their capacity to differentiate between live and dead bacteria. Prussian blue (PB) may act as a metabolic indicator, being reduced by bacterial metabolism, producing a visible color change from blue to colorless. This molecule can be present in two main forms, namely, the soluble and the insoluble, having different properties and structures. In the current work, the bacterial-sensing capacity of soluble and insoluble PB will be tested and compared both in suspensions as PB-NPs and after deposition on transparent indium tin oxide-poly(ethylene terephthalate) (ITO-PET) electrodes. In the presence of live bacteria, PB-NPs are metabolized and completely reduced to the Prussian white state in less than 10 h for soluble and insoluble forms. However, when electrodeposited on ITO-PET substrates, less than 1 h of incubation with bacteria is required for both forms, although the soluble one presents faster metabolic reduction kinetics. This study paves the way to the use of Prussian blue as a metabolic indicator for the early detection of bacterial infection in fields like microbial diagnostics, surface sterilization, food and beverage contamination, and environmental pollution, among others.

Bacterial Characteristics of Dust Particle Saltation in Gobi Dust Sites, Mongolia

The Gobi Desert is a major source of Asian dust events, and the resulting health hazards have increased significantly in recent years. We reported that a variety of live bacteria were distributed in the Gobi Desert in relation to land use. Bacterial distribution was confirmed in the environment and on the land used by animals; however, bacterial saltation due to dust events has not been investigated in detail. In this study, to understand the distribution of surface bacteria in the atmosphere by dust saltation, live bacteria in four dust-generating areas in the Gobi area were monitored using an artificial dust generating device. The live bacteria were detected by experimental saltation at a wind speed of 6.5–8 m/s in all areas. A certain number of live bacteria are constantly saltated by dust events, and these bacteria depend on land use. Moreover, the bacterial saltation strain depended on land use and diversity, indicating that live bacteria are lifted into the environment by dust events. These findings indicate that dust events saltate environmental bacteria on the ground, suggest the risk of animal-derived bacterial saltation affected by land use, and present cross-border public health challenges to be considered in the future.

Immunomodulatory receptor VSIG4 is released during spontaneous bacterial peritonitis and predicts short-term mortality

Background & AimsV-set Ig-domain-containing 4 (VSIG4) is an immunomodulatory macrophage complement receptor modulating innate and adaptive immunity and affecting the resolution of bacterial infections. Given its expression on peritoneal macrophages (PMs), we hypothesised a prognostic role of peritoneal VSIG4 concentrations in patients with spontaneous bacterial peritonitis (SBP).MethodsWe isolated PMs from patients with cirrhosis and analysed VSIG4 expression and release by flow cytometry, quantitative real-time PCR, ELISA, and confocal microscopy. We measured soluble VSIG4 concentrations in ascites from 120 patients with SBP and 40 patients without SBP and investigated the association of soluble VSIG4 in ascites with 90-day survival after SBP using Kaplan–Meier statistics, Cox regression, and competing-risks regression analysis.ResultsVSIG4 expression was high on resting, large PMs, which co-expressed CD206, CD163, and tyrosine-protein kinase Mer (MERTK). VSIG4 gene expression in PMs decreased in patients with SBP and normalised after resolution. During SBP, VSIG4hi PMs were depleted (25% vs. 57%; p <0.001) and soluble VSIG4 in ascites were higher in patients with SBP than in patients without (0.73 vs. 0.35 μg/ml; p <0.0001). PM activation by Toll-like receptor (TLR) agonists or infection with live bacteria in vitro resulted in a loss of surface VSIG4 and the release of soluble VSIG4. Mechanistically, shedding of VSIG4 from PMs was protease-dependent and susceptible to microtubule transport inhibition. Soluble VSIG4 in ascites exceeded serum concentrations and correlated with serum creatinine, model for end-stage liver disease score and C-reactive protein during SBP. Concentrations of 1.0206 μg/ml or higher indicated increased 90-day mortality (hazard ratio 1.70; 95% CI 1.01–2.86; p = 0.046).ConclusionsVSIG4 is released from activated PMs into ascites during SBP. Higher peritoneal VSIG4 levels indicate patients with organ failure and poor prognosis.Lay summaryPatients with liver cirrhosis who develop ascites have an increased risk of infection and mortality. Our study shows that in patients with infected ascites, the complement receptor VSIG4 is released by resident macrophages into the abdominal fluid where it can be measured. Patients with elevated levels of this protein in ascites are at high risk of dying within 90 days.

Novel Nematode-Killing Protein-1 (Nkp-1) from a Marine Epiphytic Bacterium Pseudoalteromonas tunicata.

Drug resistance among parasitic nematodes has resulted in an urgent need for the development of new therapies. However, the high re-discovery rate of anti-nematode compounds from terrestrial environments necessitates a new repository for future drug research. Marine epiphytes are hypothesised to produce nematicidal compounds as a defence against bacterivorous predators, thus representing a promising yet underexplored source for anti-nematode drug discovery. The marine epiphytic bacterium Pseudoalteromonas tunicata is known to produce several bioactive compounds. Screening heterologously expressed genomic libraries of P. tunicata against the nematode Caenorhabditis elegans, identified as an E. coli clone (HG8), shows fast-killing activity. Here we show that clone HG8 produces a novel nematode-killing protein-1 (Nkp-1) harbouring a predicted carbohydrate-binding domain with weak homology to known bacterial pore-forming toxins. We found bacteria expressing Nkp-1 were able to colonise the C. elegans intestine, with exposure to both live bacteria and protein extracts resulting in physical damage and necrosis, leading to nematode death within 24 h of exposure. Furthermore, this study revealed C. elegans dar (deformed anal region) and internal hatching may act as a nematode defence strategy against Nkp-1 toxicity. The characterisation of this novel protein and putative mode of action not only contributes to the development of novel anti-nematode applications in the future but reaffirms the potential of marine epiphytic bacteria as a new source of novel biomolecules.

Challenges in the Microbiological Diagnosis of Implant-Associated Infections: A Summary of the Current Knowledge.

Implant-associated infections are characterized by microbial biofilm formation on implant surface, which renders the microbiological diagnosis challenging and requires, in the majority of cases, a complete device removal along with a prolonged antimicrobial therapy. Traditional cultures have shown unsatisfactory sensitivity and a significant advance in the field has been represented by both the application of the sonication technique for the detachment of live bacteria from biofilm and the implementation of metabolic and molecular assays. However, despite the recent progresses in the microbiological diagnosis have considerably reduced the rate of culture-negative infections, still their reported incidence is not negligible. Overall, several culture- and non-culture based methods have been developed for diagnosis optimization, which mostly relies on pre-operative and intra-operative (i.e., removed implants and surrounding tissues) samples. This review outlines the principal culture- and non-culture based methods for the diagnosis of the causative agents of implant-associated infections and gives an overview on their application in the clinical practice. Furthermore, advantages and disadvantages of each method are described.

Phase separation in the outer membrane of Escherichia coli

Gram-negative bacteria are surrounded by a protective outer membrane (OM) with phospholipids in its inner leaflet and lipopolysaccharides (LPS) in its outer leaflet. The OM is also populated with many β-barrel outer-membrane proteins (OMPs), some of which have been shown to cluster into supramolecular assemblies. However, it remains unknown how abundant OMPs are organized across the entire bacterial surface and how this relates to the lipids in the membrane. Here, we reveal how the OM is organized from molecular to cellular length scales, using atomic force microscopy to visualize the OM of live bacteria, including engineered Escherichia coli strains and complemented by specific labeling of abundant OMPs. We find that a predominant OMP in the E. coli OM, the porin OmpF, forms a near-static network across the surface, which is interspersed with barren patches of LPS that grow and merge with other patches during cell elongation. Embedded within the porin network is OmpA, which forms noncovalent interactions to the underlying cell wall. When the OM is destabilized by mislocalization of phospholipids to the outer leaflet, a new phase appears, correlating with bacterial sensitivity to harsh environments. We conclude that the OM is a mosaic of phase-separated LPS-rich and OMP-rich regions, the maintenance of which is essential to the integrity of the membrane and hence to the lifestyle of a gram-negative bacterium.

Combinatorial Click Chemistry Labeling to Study Live Human Gut-Derived Microbiota Communities.

Gut bacteria were shown to exert pivotal effects on health and disease. However, mechanistic studies of gut bacterial communities are limited due to the lack of technologies for real-time studies on live bacteria. Here, we developed COMBInatorial cliCK-chemistry (COMBICK) labeling on human gut-derived bacteria, both aerobic and anaerobic strains, to enable dynamic tracing of live, heterogeneous bacterial communities on the strain level, including clinical isolates of the Enterobacteriaceae family. We further show that COMBICK labeling is applicable on anaerobic bacterial strains directly isolated from stool. In COMBICK, the number of labeled bacteria that can be simultaneously differentiated increases exponentially depending on the availability of fluorophores and machine capabilities. This method allows real-time studies of bacterial communities from a variety of ecosystems, and can significantly advance mechanistic research in the microbiome field.

Establishment of novel in vitro culture system with the ability to reproduce oral biofilm formation on dental materials

Intensive research has been conducted with the aim of developing dental restorative/prosthetic materials with antibacterial and anti-biofilm effects that contribute to controlling bacterial infection in the oral cavity. In situ evaluations were performed to assess the clinical efficacy of these materials by exposing them to oral environments. However, it is difficult to recruit many participants to collect sufficient amount of data for scientific analysis. This study aimed to assemble an original flow-cell type bioreactor equipped with two flow routes and assess its usefulness by evaluating the ability to reproduce in situ oral biofilms formed on restorative materials. A drop of bacterial suspension collected from human saliva and 0.2% sucrose solution was introduced into the assembled bioreactor while maintaining the incubation conditions. The bioreactor was able to mimic the number of bacterial cells, live/dead bacterial volume, and volume fraction of live bacteria in the in situ oral biofilm formed on the surface of restorative materials. The usefulness of the established culture system was further validated by a clear demonstration of the anti-biofilm effects of a glass-ionomer cement incorporating zinc-releasing glasses when evaluated by this system.

Live Biotherapeutic Products and Probiotics for the Skin

The skin microbiome is essential for the proper barrier functions of the skin, preventing the invasion of pathogens and regulating immune responses. When disrupted, recurrent conditions including atopic dermatitis, acne, or chronic wounds can result. Live biotherapeutic products (LBPs), an emerging class of therapeutics based on live bacteria, are promising for treating these challenging conditions. Several topical LBPs have entered clinical trials for the skin and are quickly reporting positive data from early trials. Herein, a selection of these clinical trials are analyzed to highlight the potential of topical LBPs and provide insights into their identification and development. In addition, the outstanding challenges that need to be addressed for the full translation of these LBPs into clinical therapeutics are commented. In doing so, an overview of the existing formulation landscape for microbial‐based topical products, including direct‐to‐consumer probiotics, is provided. Once the outstanding formulation and delivery challenges are addressed for LBPs and the first topical LBPs reach the market, they will be a promising modality that provides novel therapeutic mechanisms for addressing previously difficult‐to‐treat conditions.

​Ice Cream and Frozen Yoghurt - A Suitable Carrier for Probiotics: A Review

Nowadays there has been a significant interest in the development of innovative functional food products conferring customized benefits to the consumers viz., physical and mental well-being, dental health, gastro-intestinal functions, etc. Among the dairy products with live cultures, probiotic ice cream and fermented frozen desserts such as frozen yoghurt are the emerging functional foods. Ice creams and frozen yoghurt are food products showing potential for use as probiotic vehicles and are highly popular with the consumers. The problem to be tackled relates to the loss of viability of probiotic cells in frozen dairy desserts which can occur during product formulation, processing, freezing and storage. The development of probiotic frozen dairy desserts containing live probiotic bacteria necessitates certain technological interventions. The means used to attain higher survivability of probiotic cells in such frozen products include use of selected probiotic strains, use of prebiotics along with probiotics, encapsulation of probiotic cells, use of cryoprotectants, etc.

Vision redemption: Self-reporting AIEgens for combined treatment of bacterial keratitis

Bacterial keratitis (BK) is one of the most commonly leading causes of visual impairment and blindness worldwide, and suffers the risk of drug-resistant infections due to the abuse of antibiotics. Herein, we report a cationic diphenyl luminogen with aggregation-induced emission called IQ-Cm containing isoquinolinium and coumarin units for theranostic study of BK. IQ-Cm has no obvious cytotoxicity to mammalian cells below a certain concentration, and could preferentially bind to bacteria over mammalian cells. IQ-Cm can be used as a sensitive self-reporting probe to rapidly discriminate live and dead bacteria by the visual emission colors. The intrinsic dark toxicity to bacteria and generation of reactive oxygen species under light irradiation endow IQ-Cm with excellent antibacterial activity in vitro and in BK rabbit models infected with S. aureus. The present study provides a sensitive and efficient theranostic strategy for rapid discrimination of various bacterial states and the combined treatment of BK based on the intrinsic dark antibacterial activity and photodynamic therapy effect.

Evaluation of the Antioxidant Effects of Postbiotics and Paraprobiotics in Lactic Acid Bacteria Isolated from Traditional Fermented Sausages

Recent studies in the field of probiotic biotechnology have shown that some non-living microorganisms or the metabolites they produce have at least as valuable biological activities as probiotic bacteria. Paraprobiotics are described as non-viable microbial cells that confer a benefit to the human or animal when administered in adequate amounts. Bioactive soluble metabolic byproducts produced by live probiotic microorganisms that impart some physiological benefit to the host are referred to as postbiotics. Live probiotic bacteria have been used for many years to strengthen the antioxidant defense system against oxidative stress that causes many diseases. The antioxidant activity of postbiotics and paraprobiotics in lactic acid bacteria isolated from twelve different hand-made fermented sausages was assessed using the DPPH radical scavenging assay in this research. It was determined that the free radical scavenging effect of postbiotics used in the study was between 5.65% and 76.04%, and the effect of paraprobiotics varied between 5.90% and 18.07%. According to the findings, antioxidant capacity of the postbiotics and paraprobiotics are strain-dependent and the postbiotics have higher antioxidant activity than the paraprobiotics.

Safety Assessment of a New Strain of Lactobacillus pentosus (IBRC=11143) As a Candidate Probiotic

Background: It is believed that the consumption of an adequate amount of live lactic acid bacteria (as probiotic) may improve the health of the host. Many strains of lactic acid bacteria are generally considered to be safe. However, some strains have shown some adverse effects.&#x0D; Objectives: This study aimed to investigate the safety of a new strain of Lactobacillus pentosus (IBRC=11143) in Wistar rats&#x0D; Methods:In this experimental study, male and female Wistar rats (n=6) were used. A subacute toxicity study (for 28 days) was conducted by oral administration of Lactobacillus pentosus to the animals. In each sex, one group received saline, and the other two groups received the bacteria at doses of 1×108 and 1×109 Colony-Forming Units (CFU)/rat.&#x0D; Results: No significant alteration in the liver and kidney tissues was seen. However, in both sexes, there were significant differences in urea and creatinine levels between the control and the experimental groups. Some blood parameters (Lymphocyte, red blood cell, hematocrit, and hemoglobin) also showed significant changes in the groups that received the bacteria. Moreover, a significant increase in alkaline phosphatase level was observed in male rats.&#x0D; Conclusion: The results indicate that Lactobacillus pentosus (11143) is not entirely safe like other Lactobacillus strains. Therefore, the Lactobacillus pentosus (11143) strain may not be the right choice as a probiotic for human consumption.

β-Glucan-triggered Akkermansia muciniphila expansion facilitates the expulsion of intestinal helminth via TLR2 in mice

Trichinellosis caused by Trichinella spiralis is a serious zoonosis with a worldwide. β-Glucans (BG) are readily used across the world with noted health benefits, yet the effect and mechanism of BG on host defense against helminth infection remain poorly understood. We observed that BG could trigger worm expulsion via mucus layer independently of type 2 immunity, but was dependent on the gut microbiota in mice. BG restored the abundance of Bacteroidetes and Proteobacteria changed by T. spiralis infection to the control group level and markedly increased the relative abundance of Verrucomicrobia. Akkermansia (belonging to Verrucomicrobia) were significantly expanded in the BG + T. spiralis group. Notably, daily oral supplementation of pasteurized A. muciniphila has a stronger deworming effect than live bacteria and interacted with TLR2. These findings of this study is an easily implementable strategy to facilitate expulsion of gastrointestinal helminth.

Antibody Mimics as Bio-orthogonal Catalysts for Highly Selective Bacterial Recognition and Antimicrobial Therapy.

Bacterial infectious diseases seriously threaten public health and life. The specific interaction between an antibody and its multivalent antigen is an attractive way to defeat infectious disease. However, due to the high expense and strict storage and applied conditions for antibodies, it is highly desirable but remains an urgent challenge for disease diagnosis and treatment to construct artificial antibodies with strong stability and binding ability and excellent selectivity. Herein, we designed and synthesized antibody-like bio-orthogonal catalysts with the ability to recognize specific bacteria and accomplish in situ drug synthesis in captured bacteria by using improved bacterial imprinting technology. On one hand, the artificial antibody possesses a matching morphology for binding pathogens, and on the other hand, it acts as a bio-orthogonal catalyst for in situ synthesis of antibacterial drugs in live bacteria. Both in vitro and in vivo experiments have demonstrated that our designed antibody can distinguish and selectively bind to specific pathogens and eliminate them on site with the activated drugs. Therefore, our work provides a strategy for designing artificial antibodies with bio-orthogonal catalytic activity and may broaden the application of bio-orthogonal chemistry.

Bismuth phosphinato incorporated antibacterial filter paper for drinking water disinfection

Lack of safe drinking water is a major concern, mainly in developing countries. Consequently, there is a significant need for affordable technologies to provide clean drinking water. In this paper, two approaches for drinking water disinfection using nanocellulose and the antimicrobial compound phenyl bis -diphenylphosphinato bismuth (III) complex are studied. The first approach investigates the deactivation of bacteria when contaminated water percolates through a filter paper with a microfibrillated cellulose (MFC) coating containing Bi-complex. Antibacterial activity was studied by filtering bacterial broth with a Gram-positive pathogen Staphylococcus aureus as a test strain and measuring live bacteria in the filtrate. The filter composites did not show any sign of instantaneous killing of bacteria, irrespective of the Bi-complex loading or base paper substrate. However, the effluent demonstrated drastic reduction in bacterial population after resting at 37 °C. Bacteria were completely eradicated 24 h after passing through 5 wt% and 10 wt% Bi-coated filters. If also active against locally important drinking water contaminants, this approach could be used for purification of storage water. The second approach uses a Bi-complex loaded nanofibrillated cellulose (NFC) membrane. Herein, complete and immediate exclusion of bacteria was achieved in the NFC membrane when bacterial broth was passed through it at 1 bar. The 2.5 wt% Bi-loaded membrane could significantly minimise the risk of biofilm formation, even after 4 days of contact with bacterial broth. The Bi-complex concentration in the filtrate (130.6 ± 1.9 ppb) from the 2.5 wt% Bi-loaded microfilter was shown to be non-toxic to murine fibroblast cells. This study outlines the proof of concept of two sustainable prototypes for stored or immediate drinking water purification, but the approach would need to be extended to a broader range of relevant contaminating pathogens of drinking water to fully understand its utility. • Bi-complex coated filters show no instantaneous reduction in bacterial population. • Bi-complex leaches into the filtrate killing bacteria after storage at 37 °C. • Complete exclusion of bacteria achieved by nanofibrillated cellulose (NFC) membrane. • Bi-complex in the NFC membranes minimises the risks of biofilm formation. • In-vitro studies shows the Bi-complex at or below 200 ppb is not cytotoxic.

The myth of antibiotic spider silk

SummarySpider silk is frequently attributed antimicrobial properties. This notion is based on studies reporting antimicrobial activity (AMA) of spider silk; however, close inspection of these studies reveals that the evidence is conflicting, and at best anecdotal. We performed a systematic study of antimicrobial properties of different silk types from seven species across the spider phylogeny. We found no evidence of AMA of silk in direct contact and disc diffusion assays against Gram-negative Escherichia coli and Pseudomonas putida, and the Gram-positive Bacillus subtilis. Furthermore, staining experiments and fluorescence microscopy showed the presence of live bacteria on silk surfaces indicating no antimicrobial effect on direct contact. A critical evaluation of the literature reveals that published tests of AMA are scarce and that all the studies claiming positive results are compromised by methodological shortcomings. Our analysis demonstrates that the common notion that spider silk is antimicrobial is not supported by empirical data.

VEGF-C and podoplanin, as biomarkers of sepsis. An experimental study

Abstract Background: Sepsis is the leading cause of morbidity and mortality in intensive care units. This study explored the possible role of vascular endothelial growth factor-C (VEGF-C) and podoplanin (PDPN) in sepsis. Methods: 22 Wistar rats were divided into three groups: two experimental (Group A and B, n=8/8) and a control (Group C, n=6). Sepsis was induced with intraperitoneal injection of ESBL (extended-spectrum beta-lactamases)-producing E-coli live bacteria for group A and with lipopolysaccharide for group B. Sterile saline solution was injected for group C. Blood samples were collected after 24 hours to determine the serum level of VEGF-C, and PDPN expression was examined in liver, kidney, and lung tissues. Bacteremia was assessed for group A. Results: Higher serum levels of VEGF-C were found in Group A vs C (p=0.05) and group B vs. C (p=0.004), respectively.VEGF-C was also increased in animals with negative- vs. positive blood cultures from group A (p=0.04) and from group B vs. those with positive blood cultures from group A (p=0.03). High intensity of PDPN tissue expression was observed in the pulmonary alveolocytes from Group A and epithelium of the proximal renal tubules in groups B and C, compared to group A. Conclusions: Circulating VEGF-C can be succesfuly used as a biomarker of sepsis with negative blood cultures and high risk of renal failure, whereas PDPN seems to exert a protective role against lung injuries in live bacteria-induced sepsis.

Effects of Live and Pasteurized Forms of Akkermansia from the Human Gut on Obesity and Metabolic Dysregulation.

Akkermansia muciniphila (A. muciniphila) is a promising probiotic candidate owing to its health-promoting properties. A previous study reported that the pasteurized form of A. muciniphila strains isolated from human stool samples had a beneficial impact on high-fat diet-induced obese mice. On the other hand, the differences in the probiotic effects between live and pasteurized A. muciniphila on the metabolism and immune system of the host are still inconclusive. This study examines the differences between the live and pasteurized forms of A. muciniphila strains on the lipid and glucose metabolism and on regulating the inflammatory immune responses using a HFD-fed obese mouse model. The animals were administered the live and pasteurized forms of two A. muciniphila strains five times per week for the entire study period of 12 weeks. Both forms of the bacterial strains improved the HFD-induced obesity and metabolic dysregulation in the mice by preventing body-weight gains after one week. In addition, they cause a decrease in the weights of the major adipose tissues, adipogenesis/lipogenesis and serum TC levels, improvement in glucose homeostasis and suppression of inflammatory insults. Furthermore, these treatments restored the damaged gut architecture and integrity and improved the hepatic structure and function in HFD-induced animals. On the other hand, for both bacterial strains, the pasteurized form was more potent in improving glucose tolerance than the live form. Moreover, specific A. muciniphila preparations with either live or pasteurized bacteria decreased the number and population (%) of splenic Treg cells (CD4+ Foxp3+) significantly in the HFD-fed animals, further supporting the anti-inflammatory properties of these bacteria.

Comparison of Two Cutibacterium acnes Biofilm Models.

The study of biofilms in vitro is complex and often limited by technical problems due to simplified models. Here, we compared C. acnes biofilm formation, from species involved in bone and prosthesis infection, in a static model with a dynamic model. Using similar parameters, the percentage of live bacteria within the biofilm was higher in dynamic than in static approach. In both models, bacterial internalization in osteoblast-like cells, playing the role of stress factor, affected this proportion but in opposite ways: increase of live bacteria proportion in the static model (×2.04 ± 0.53) and of dead bacteria proportion (×3.5 ± 1.03) in the dynamic model. This work highlights the huge importance in the selection of a relevant biofilm model in accordance with the environmental or clinical context to effectively improve the understanding of biofilms and the development of better antibiofilm strategies.