Microbial Sensor Research Articles

Chitosan matrices modified with carbon nanotubes for use in mediated microbial biosensing

We describe a microbial sensor based on Pseudomonas fluorescens cells that was prepared by modifying graphite electrodes with chitosan and carbon nanotubes. Chronoamperometry was performed at +0.3 V in the presence of hexacyanoferrate as a mediator and revealed a good response to glucose which is linear in the 1.0 to 5.0 mM concentration range. Linearity was defined by the equation of y = 102.120x−13.279 (R 2 = 0.998) (y shows current density as nA.cm−2 and x shows glucose concentration in mM). The effect of the CNTs on the response was compared to that of electrodes made without CNTs.

Integrated MOSFET-Embedded-Cantilever-Based Biosensor Characteristic for Detection of Anthrax Simulant

In this work, MOSFET-embedded cantilevers are configured as microbial sensors for detection of anthrax simulants, Bacillus thuringiensis. Anthrax simulants attached to the chemically treated gold-coated cantilever cause changes in the MOSFET drain current due to the bending of the cantilever which indicates the detection of anthrax simulant. Electrical properties of the anthrax simulant are also responsible for the change in the drain current. The test results suggest a detection range of 10 μL of stimulant test solution (a suspension population of 1.3 × 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">7</sup> colony-forming units/mL diluted in 40% ethanol and 60% deionized water) with a linear response of 31 μA/μL.

DC-SIGN Mediated Sphingomyelinase-Activation and Ceramide Generation Is Essential for Enhancement of Viral Uptake in Dendritic Cells

As pattern recognition receptor on dendritic cells (DCs), DC-SIGN binds carbohydrate structures on its pathogen ligands and essentially determines host pathogen interactions because it both skews T cell responses and enhances pathogen uptake for cis infection and/or T cell trans-infection. How these processes are initiated at the plasma membrane level is poorly understood. We now show that DC-SIGN ligation on DCs by antibodies, mannan or measles virus (MV) causes rapid activation of neutral and acid sphingomyelinases followed by accumulation of ceramides in the outer membrane leaflet. SMase activation is important in promoting DC-SIGN signaling, but also for enhancement of MV uptake into DCs. DC-SIGN-dependent SMase activation induces efficient, transient recruitment of CD150, which functions both as MV uptake receptor and microbial sensor, from an intracellular Lamp-1+ storage compartment shared with acid sphingomyelinase (ASM) within a few minutes. Subsequently, CD150 is displayed at the cell surface and co-clusters with DC-SIGN. Thus, DC-SIGN ligation initiates SMase-dependent formation of ceramide-enriched membrane microdomains which promote vertical segregation of CD150 from intracellular storage compartments along with ASM. Given the ability to promote receptor and signalosome co-segration into (or exclusion from) ceramide enriched microdomains which provide a favorable environment for membrane fusion, DC-SIGN-dependent SMase activation may be of general importance for modes and efficiency of pathogen uptake into DCs, and their routing to specific compartments, but also for modulating T cell responses.

A Novel Design of BOD Biosensor Based on Immobilized Microbial Biosensor

In this study, an instrument for circulating BOD analysis by microbial sensor has been developed. A Pseudomonas. sp immobilized by PVA, was employed for sensitive material BOD sensor. The measure period was about 8 minutes. Both relative error and analytical standard deviation are less than 5%. The instrument has the advantages of high autoimmunization and short measure period. The accuracy and stability of the instrument measured up the national standard by testing different type waste waters and national standard samples.

Immune Evasion Tactic

A viral homolog of a host microbial sensor inhibits viral detection by the host and subsequent antiviral immunity.

Microbial biosensors for detection of biological oxygen demand (a Review)

The review briefs recent advances in application of biosensors for determining biological oxygen demand (BOD) in water. Special attention is focused on the principles of operation of microbial BOD sensors; the information about biorecognition elements in such systems and the methods used for immobilization of biological components in film biosensors is summarized. Characteristics of some BOD sensor models are considered in detail.

An electrochemical impedance model for integrated bacterial biofilms

Bacterial cells attachment onto solid surfaces and the following growth into mature microbial biofilms may result in highly antibiotic resistant biofilms. Such biofilms may be incidentally formed on tissues or implanted devices, or intentionally formed by directed deposition of microbial sensors on whole-cell bio-chip surface. A new method for electrical characterization of the later on-chip microbial biofilm buildup is presented in this paper. Measurement of impedance vs. frequency in the range of 100 mHz to 400 kHz of Escherichia coli cells attachment to indium-tin-oxide-coated electrodes was carried out while using optical microscopy estimating the electrode area coverage. We show that impedance spectroscopy measurements can be interpreted by a simple electrical equivalent model characterizing both attachment and growth of the biofilm. The correlation of extracted equivalent electrical lumped components with the visual biofilm parameters and their dependence on the attachment and growth phases is confirmed.

Abstract ED06-04: The microbiota and development of colitis-associated colorectal cancer

Abstract The human intestinal microbiota is a community of 1013-1014 microorganisms that reside in the intestine and normally participate in a symbiotic relationship with their eukaryotic host. The microbiome contains a wealth of information, encompassing 150 fold as many genes as the human genome. Recent studies have hinted that a core human microbiome exists at the gene level, with a large number of microbial genes and pathways shared among individuals. Deviations from this core microbiome could potentially affect human health and promote disease state. Thus characterizing the vast intestinal microbial community present in healthy as well as pathologic conditions will likely revolutionize our conception of bacteria/host interactions and identify new physiological processes influenced by the microbiota. Already the microbiota has been linked to cardiac development, angiogenesis, innate and adaptive immunity, metabolism, nutrient acquisition, and gastrointestinal development and homeostasis. Furthermore, alterations in the microbial community are associated with multiple diseases, including obesity, fatty liver disease, type 1 and type 2 diabetes, kidney disease, arthritis and inflammatory bowel diseases (IBD). Eukaryotes are equipped with an elegant repertoire of innate receptors each recognizing specific conserved microbial patterns present on various microorganisms, such as components of bacterial cell walls, locomotion system or nucleic acids. These microbial sensors are termed pattern recognition receptors (PRR) and include retinoic acid inducible gene-I like RNA helicases (RLH), C-type lectin receptors (CLR), Nucleotide-binding domain leucine-rich repeat proteins (NLR; also known as Nod-like receptors), and Toll-like receptors (TLR). Although the etiology of colitis-associated colorectal cancer remains elusive, innate host sensors have been shown to modulate the pathogenesis. Indeed, using mice exposed to the colon-specific carcinogen azoxymethane (AOM) compound and to the mucosal-damaging agent dextran sulfate sodium (DSS), numerous studies have revealed the differential contribution of innate sensors to CAC. For example, the NLR protein NOD1, a close relative of the IBD susceptible gene NOD2 protects against the development of colitis and colorectal cancer. Similarly, the adaptor protein MyD88 frequently utilized by TLR family members is protective in the context of AOM/DSS-induced colorectal cancer. Furthermore, the intracellular sensor NLRP3 also appears protective against colitis and CAC. In contrast, TLR4 signaling promotes the development of colorectal cancer in the AOM/DSS model. Although informative, these studies only capture the impact of various innate host responses on the development of colorectal cancer. The true impact of the microbiota could only be investigated using germ-free mice and gnotobiotic technology. To avoid the use of a chronic injury model (DSS), our laboratory has substituted the spontaneous IL-10−/− mouse model of intestinal inflammation for the AOM/DSS model. We have focused our attention on the role of microbial community on the development of colitis-associated colorectal cancer. Using this AOM/Il10−/− model of colorectal cancer in combination with gnotobiotic techniques, we observed a clear role for the microbiota in the development and progression of the pathology. Using metagenomic approaches, we observed a shift in bacterial community composition in mice bearing tumors compared to healthy controls. Interestingly, introduction of the probiotic VSL#3 changed microbial community structure and promoted the development of colorectal cancer in Il10−/− mice. Gnotobiotic experiments showed that bacteria-induced intestinal inflammation is not sufficient to promote colorectal cancer. Indeed, although the severity of colitis at the clinical and molecular levels is similar between Escherichia coli NC101 (E. coli) and Enterococcus faecalis (E. faecalis) monoassociated Il10−/− mice, tumor numbers were enhanced by more than 40 fold by the former commensal bacterium. We will present evidence that microbial composition and its associated genome/transcriptome plays a key role in the development of CAC. These findings would suggest that although chronic inflammation is a part of the pathology, microbial community and associated microbial genome is at the forefront of CAC susceptibility. Altering the composition of the microbiota may represent a novel means by which to modulate development of colorectal cancer in IBD patients. Citation Information: Cancer Prev Res 2010;3(12 Suppl):ED06-04.

SLAM is a microbial sensor that regulates bacterial phagosome functions in macrophages

Phagocytosis is a pivotal process by which macrophages eliminate microorganisms after recognition by pathogen sensors. Here we unexpectedly found that the self ligand and cell surface receptor SLAM functioned not only as a costimulatory molecule but also as a microbial sensor that controlled the killing of gram-negative bacteria by macrophages. SLAM regulated activity of the NADPH oxidase NOX2 complex and phagolysosomal maturation after entering the phagosome, following interaction with the bacterial outer membrane proteins OmpC and OmpF. SLAM recruited a complex containing the intracellular class III phosphatidylinositol kinase Vps34, its regulatory protein kinase Vps15 and the autophagy-associated molecule beclin-1 to the phagosome, which was responsible for inducing the accumulation of phosphatidylinositol-3-phosphate, a regulator of both NOX2 function and phagosomal or endosomal fusion. Thus, SLAM connects the gram-negative bacterial phagosome to ubiquitous cellular machinery responsible for the control of bacterial killing.

Role of Postnatal Acquisition of the Intestinal Microbiome in the Early Development of Immune Function

Therapy with broad-spectrum antibiotics is a common practice for premature infants. This treatment can reduce the biodiversity of the fecal microbiota and may be a factor in the cause of necrotizing enterocolitis. In contrast, probiotic treatment of premature infants reduces the incidence of necrotizing enterocolitis. We hypothesized that 1 mechanism for these observations is the influence of bacteria on postnatal development of the mucosal immune system. Expression of immune molecules and microbial sensors was investigated in the postnatal mouse gastrointestinal tract by real-time polymerase chain reaction. Subsequently, 2-week-old specific pathogen-free and microbial-reduced (MR; antibiotic treated) mice were compared for immune molecule and microbial sensor expression, mesenteric lymph node T-cell numbers and activation, intestinal barrier function/permeability, systemic lymphocyte numbers, and T-cell phenotype commitment. Toll-like receptor 2, 4, and 5 expression was highest in 2-week-old specific pathogen-free mice, and this expression was decreased in MR mice. There was no difference in intestinal tight-junctional function, as evaluated by fluorescein isothiocyanate-dextran uptake, but MR mice had increased bacterial translocation across the intestinal epithelial barrier. MR mice had significantly fewer splenic B cells and mesenteric lymph node CD4+ T cells, but there were normal numbers of splenic T cells. These systemic T cells from MR mice produced more interleukin-4 and less interferon-gamma and IL-17, indicative of maintenance of the fetal, T-helper cell type 2 phenotype. The present study shows that intestinal commensal microbiota have an influence on early postnatal immune development. Determining specific bacteria and/or bacterial ligands critical for this development could provide insight into the mechanisms by which broad-spectrum antibiotics and/or probiotic therapy influence the development of the mucosal immune system and mucosal-related diseases.

NOD2polymorphisms in clinical phenotypes of common variable immunodeficiency disorders

Common variable immunodeficiency disorders (CVIDs) are a heterogeneous group of diseases characterized by hypogammaglobulinaemia and consequent susceptibility to infection. CVID patients commonly develop a variety of additional manifestations for which the causative factors are not fully understood. Two such manifestations are granulomatous disease and enteropathy. Because the ability to predict complications would aid clinical management, we continue to search for possible disease modifier genes. NOD2 acts a microbial sensor and is involved in proinflammatory signalling. Particular mutations of the NOD2 gene are associated with Crohn's disease including gly908arg, leu1007finsc and arg702trp polymorphisms. We hypothesized that NOD2 polymorphisms may be a disease modifier gene towards an enteropathic or granulomatous phenotype within CVIDs. Sequence-specific primers returned genotypes for 285 CVID patients from centres across the United Kingdom and Europe. We present the frequencies of the different phenotypes of patients within our international cohort. Arg702trp polymorphisms were significantly less frequent than wild-type (WT) (P = 0·038) among international CVID patients with splenomegaly. Gly908arg polymorphisms were more prevalent than WT in UK patients with autoimmune disorders (P = 0·049) or enteropathy (P = 0·049). NOD2 polymorphisms were not more prevalent than WT in CVID patients with clinical phenotypes of granulomata. UK allele frequencies of 0·014, 0·056 and 0·026 were found for gly908arg, arg702trp and leu1007finsc NOD2 polymorphisms, respectively. These do not differ significantly from UK immunocompetent controls confirming, as expected, that in addition these NOD2 polymorphisms do not confer susceptibility to CVIDs per se.

Inflammasomes as microbial sensors

Members of the Nod-like receptor family and the adaptor ASC assemble into multiprotein platforms, termed inflammasomes, to mediate the activation of caspase-1 and subsequent secretion of IL-1beta and IL-18. Recent studies have identified microbial and endogenous molecules as well as possible mechanisms involved in inflammasome activation.

Microbiota in Chronic Mucosal Inflammation

The human gastrointestinal tract is occupied by a complex and abundant microbial community reaching as high as 1013–1014 microorganisms in the colon. This microbiota participates in a symbiotic relationship with their eukaryotic host, and this partnership is viewed as essential for maintaining homeostasis. The microbiome contains a wealth of information, encompassing 150-fold as many genes as the human genome and performing essential and nonredundant tasks (e.g., nutrition/energy balance, immune system toning, and pathogen exclusion) for the host. Recent studies have hinted that a core human microbiome exists at the gene level, with a large number of microbial genes and pathways shared among individuals. Deviations from this core microbiome could potentially affect human health and promote disease state. The coexistence of the host with its intestinal microbiota is tightly controlled at various levels and an accumulating body of evidence suggests that the failure of this homeostasis is an important contribution to disease development. Recent discoveries clearly suggest that the gut microbiota of individuals throughout their lifespan is a powerful determinant of chronic diseases and that the mechanisms underlying this link involve the development of inflammatory activity. The microbiota has already been linked to cardiac development, angiogenesis, innate and adaptive immunity, metabolism, nutrient acquisition, and gastrointestinal development and homeostasis. Furthermore, alterations in microbial community composition are associated with multiple diseases, including obesity, fatty liver disease, type 1 and type 2 diabetes, kidney disease, arthritis, and inflammatory bowel diseases (IBD). On the host side, a series of mechanisms help contain the formidable antigenic power of the microbiota. One of the main mechanisms, which could be regrouped under the wide umbrella of functional mucosal barrier, includes the formation of epithelial tight junction, mucus production, antimicrobial peptide secretion, and immunoglobulin A release. Equally important is the presence of a sophisticated repertoire of innate receptors, each recognizing specific conserved microbial patterns present on various microorganisms. Because the basic structure of bacteria is relatively conserved, eukaryotes have developed throughout evolution sensing systems to detect these bacterial signatures which include cell wall components, locomotion system, and nucleic acids. These microbial sensors are termed pattern recognition receptors (PRR-) and include retinoic acid inducible gene-I-like RNA helicases (RLH), C-type lectin receptors (CLR), nucleotide-binding domain leucine-rich repeat proteins (NLR), also known as Nod-like receptors, and Toll-like receptors (TLR). This arsenal of innate sensors plays a critical role in maintaining intestinal homeostasis through elimination of pathogenic microorganisms and preventing the formation of a dysbiotic microbiota. Interestingly, modulation of the gut microbiota and prevention of a dysbiotic state have become the focus of intense research. Indeed, various strategies including the introduction of prebiotic, probiotic, dietary compounds, and recently fecal transplantation to treat recurrent C. difficile infection have received special attention to treat various intestinal inflammatory disorders. Clearly, one could appreciate the complexity of these host-microbe interaction and the deleterious consequences of an improper response to both the microbiota and infectious microorganisms. For example, Nod2 polymorph-isms causing a loss of bacterial recognition mechanisms have been identified as susceptibility factors for Crohn's disease. Moreover, NOD2 variants are also implicated in the development of gingivitis and Graft-versus-host disease after bone marrow transplantation. This special issue of the International Journal of Inflammation brings into focus the complexity of bacteria-host interactions and the impact of these interactions on intestinal homeostasis, and conversely in the development of various pathological conditions. Gerhard Rogler Dirk Haller Christian Jobin

Microbial Biosensors for Selective Detection of Disaccharides

Seven microbial strains were screened for their ability to detect disaccharides as components of Clark-type oxygen biosensors. Sensors responded to varying degrees to maltose, cellobiose, sucrose, and melibiose, but none responded strongly to lactose. Although microbial sensors are relatively nonspecific, it is possible to obtain differential measurements of specific substrates using multiple sensors with different relative specificities. For example, Escherichia coli strain K-802 oxidized maltose but had low activity against sucrose, while Bacillus subtilis strain VKM B-434 responded more strongly to sucrose than maltose. Furthermore, signals from these two sensors were additive for selected samples. Results suggest that a two-component biosensor utilizing these strains could be used for differential detection of sucrose and maltose.

NOD2 contributes to cutaneous defense againstStaphylococcus aureusthrough α-toxin-dependent innate immune activation

Staphylococcus aureus is a major cause of community-acquired and nosocomial infections including the life-threatening conditions endocarditis, necrotizing pneumonia, necrotizing fasciitis, and septicemia. Toll-like receptor (TLR)-2, a membrane-bound microbial sensor, detects staphylococcal components, but macrophages lacking TLR2 or both TLR2 and TLR4 remain S. aureus responsive, suggesting that an alternative microbial recognition receptor might be involved. The cytoplasmic sensor nucleotide-binding oligomerization domain containing (NOD) 2/caspase recruitment domain (CARD) 15 detects muramyl dipeptide from bacterial peptidoglycans and mediates cytokine responses to S. aureus in vitro, but the physiological significance of these observations is not well defined. Here we show that NOD2-deficient mice exhibit a delayed but ultimately exacerbated ulcerative response and impaired bacterial clearance after s.c. infection with S. aureus. NOD2-dependent recognition of S. aureus and muramyl dipeptide is facilitated by alpha-toxin (alpha-hemolysin), a pore-forming toxin and virulence factor of the pathogen. The action of NOD2 is dependent on IL-1beta-amplified production of IL-6, which promotes rapid bacterial killing by neutrophils. These results significantly broaden the physiological importance of NOD2 in innate immunity from the recognition of bacteria that primarily enter the cytoplasm to the detection of bacteria that typically reside extracellularly and demonstrate that this microbial sensor contributes to the discrimination between commensal bacteria and bacterial pathogens that elaborate pore-forming toxins.

Online monitoring of yeast cultivation using a fuel-cell-type activity sensor

A microbial fuel-cell type activity sensor integrated into 500 mL and 3.2 L bioreactors was employed for ampero- (lA) and potentiometric (mV) measurements. The aim was to follow the microbial activity during ethanol production by Saccharomyces cerevisiae and to detect the end of carbohydrate consumption. Three different sensor setups were tested to record electrochemical signals produced by the metabolism of glucose and fructose (1:1) online. In a first setup, a reference electrode was used to record the potentiometric values, which rose from 0.26 to 0.5 V in about 10 h during the growth phase. In a second setup, a combination of ampero- and pseudo-potentiometric measurements delivered a maximum voltage of 35 mV. In this arrangement, the pseudo-potentiometric signal changed in a manner that was directly proportional to the amperometric signals, which reached a maximum value of 32 muA. In a third type of arrangement, a reference electrode was added to the anodic bioreactor compartment to carry out ampero- and potentiometric measurements; this is made possible by the high internal resistance of the cultivation. In this case, the reference potential rose to 0.44 V while the current maximum recorded by the working electrodes reached 27 lA. Reference and pseudo-reference electrodes were in all cases K(3)Fe(CN)(6)/carbon. Electrodes were made of 9 cm(2) woven graphite. To compare the electrochemical signals with established values, the metabolism was also monitored for optical density (at 600 nm) indicating biomass production. For fructose and glucose conversion, HPLC with an Aminex column and RI detector was used, and ethanol production was analyzed by GC with methanol as internal standard. The combination of amperometric and potentiometric recordings was found to be an ideal setup and was successfully used in reproducible cultivations.

A Microbial Sensor Based on Direct Electron Transfer at Shewanella Sp. Drop‐Coated Screen‐Printed Carbon Electrodes

AbstractAerobically grown Shewanella sp. bacterial suspension drop‐coated on a disposable screen‐printed carbon electrode was found to possess electroactivity without the aid of redox mediator. Cyclic voltammetric studies revealed the characteristics of a mixed diffusion adsorption‐controlled electrochemical process for direct electron transfer at the bacteria‐modified electrode. Both FE‐SEM and ATR FT‐IR experiments were carried out to investigate the surface characteristics. The electroanalytical applicability was further demonstrated for electrocatalytic reduction of arsenite, hydrogen peroxide and nitrite. Low cost and very simple manufacturing procedure allow for the proposed bacterial sensor to be applied as disposable devices.

Different calibration methods of a microbial BOD sensor for analysis of municipal wastewaters

On the base of diffusion limited amperometric oxygen sensor (the Clark-type oxygen sensor) a microbial BOD sensor was constructed and the dependence of output signal of device on wastewater biochemical oxygen demand (BOD) was studied. Calibration of BOD sensor was performed in steady-state and dynamic regime of measurements in OECD synthetic wastewater solutions. Results showed broader detection area (up to 230mgBOD7L−1) as well as shorter response and regeneration times in dynamic regime of analysis of output signal. The data acquired by steady-state method showed better repeatability, especially in the estimation of BOD7 of real wastewaters. After calibration the BOD sensor was used for quick BOD estimations of municipal wastewaters.

Toxicity of nanoparticles of ZnO, CuO and TiO 2 to yeast Saccharomyces cerevisiae

The aim of this study was to evaluate the toxic effect of nanosized ZnO, CuO and TiO 2 to Saccharomyces cerevisiae – a widely used unicellular eukaryotic model organisms in molecular and cell biology . The effect of metal oxide nanoparticles, their bulk forms and respective ionic forms were compared. The bioavailable Zn 2+ and Cu 2+ ions in the growth medium were quantified by recombinant microbial sensors. Nano and bulk TiO 2 were not toxic even at 20000 mg/l. Both, nano and bulk ZnO were of comparable toxicity (8-h EC 50 121–134 mg ZnO/l and 24-h EC 50 131–158 mg/l). The toxicity was explained by soluble Zn-ions as proved by the microbial sensor. However, nano CuO was about 60-fold more toxic than bulk CuO: 8-h EC 50 were 20.7 and 1297 mg CuO/l and 24-h EC 50 were 13.4 and 873 mg/l, respectively. The increase in toxicity of both CuO formulations at 24th hour of growth was due to the increased dissolution of copper ions from CuO over time. Comparison of EC 50 values of nano CuO, bulk CuO and Cu 2+ with bioavailable copper concentrations in the growth medium showed that the solubilized Cu-ions explained only about 50% of the toxicity of both, nano and bulk CuO. To our knowledge, this is the first study that evaluates the toxicity of ZnO, CuO and TiO 2 nanoparticles to S. cerevisiae.

NOD2-associated diseases: Bridging innate immunity and autoinflammation

NOD2 is an intracellular microbial sensor of the innate immune system that can act as a potent activator and regulator of inflammation. Mutations in the gene encoding NOD2 in humans have been associated with Crohn's disease (CD), Blau syndrome (BS), and early onset sarcoidosis (EOS). These diseases have in common features of dysregulated inflammation, but have very distinct phenotypes, which have been hypothesized to result from either loss-of-function (CD) or gain-of-function (BS/EOS) mutations. Here we describe an infant with early onset sarcoidosis who presented with systemic inflammation and disseminated granulomatous disease, including the triad of granulomatous arthritis, uveitis and dermatitis, as well as unusual gastrointestinal tract granulomas. The patient had a susceptibility polymorphism of NOD2 previously described in CD, but not in BS or EOS. We discuss the complex role of NOD2 in innate immunity to microbes and the clinical consequences of disturbances in this system.