Characterisation of electrochemical immunosensor for detection of viable not-culturable forms of Legionellla pneumophila in water samples

2015 ◽  
Vol 69 (11) ◽  
Author(s):  
Dejla Sboui ◽  
Mina Souiri ◽  
Stephanie Reynaud ◽  
Sabine Palle ◽  
Manel Ben Ismail ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Indium Tin Oxide ◽  
Membrane Filtration ◽  
Laser Scanning ◽  
Electrochemical Impedance ◽  
Limit Of Detection ◽  
Angle Measurement ◽  
Electrochemical Immunosensor ◽  
Laser Scanning Microscopy ◽  
Confocal Laser

AbstractLegionella pneumophila may cause a fatal pneumonia in humans known as Legionnaires’ disease (LD). The strategies of L. pneumophila to adapt to and resist stressful environmental conditions include the ability to enter into a VBNC (viable but not culturable) state. The detection of L. pneumophila in environmental samples benefits from the use of standardised methods: for detection and enumeration following membrane filtration (AFNOR T90-431, ISO 11731) and detection and quantification by polymerase chain reaction PCR (AFNOR T90-471, ISO 12869). Culture is hampered by its inability to detect VBNC forms and PCR is unable to discriminate between live and dead bacteria. The present immunosensor was obtained by the immobilisation of a monoclonal anti-L. pneumophila antibody (MAb) on an indium-tin oxide (ITO) electrode by the self-assembled monolayers (SAMs) method using an aminosilane. The immunosensor was characterised by wettability (contact angle measurement), atomic force microscopy (AFM), confocal laser scanning microscopy (CLSM), and electrochemical impedance spectroscopy (EIS). A limit of detection of 10 bacteria per mL was observed on artificial samples.

Influence of the Electrolyte Composition on the Corrosion Behavior of Anodized AZ31B Magnesium Alloy

2020 ◽  
Vol 1012 ◽  
pp. 424-429
Author(s):  
Leandro Antonio de Oliveira ◽  
Renato Altobelli Antunes
Keyword(s):  
Magnesium Alloy ◽  
Laser Scanning ◽  
Electrochemical Impedance ◽  
Electrolyte Composition ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Mixed Solution ◽  
Protective Properties ◽  
Az31b Magnesium Alloy ◽  
Substrate Dissolution

Investigations have been performed to study the effects of the electrolyte composition on the properties of anodized films grown on AZ31B magnesium alloy. The corrosion protection ability of the oxide layers was explored by using potentiodynamic polarization (PDP) and electrochemical impedance spectroscopy. Film morphology was examined by scanning electron microscopy and confocal laser scanning microscopy. In spite of its higher roughness average, the film formed in the silicate and hydroxide mixed solution enhanced the protective properties of the anodized layer, thus reducing the substrate dissolution rate.

scholarly journals HtrA Homologue of Legionella pneumophila: an Indispensable Element for Intracellular Infection of Mammalian but Not Protozoan Cells

2001 ◽  
Vol 69 (4) ◽  
pp. 2569-2579 ◽  
Author(s):  
Lisa L. Pedersen ◽  
Marina Radulic ◽  
Miljenko Doric ◽  
Yousef Abu Kwaik
Keyword(s):  
Epithelial Cells ◽  
Confocal Laser Scanning Microscopy ◽  
Legionella Pneumophila ◽  
Parental Strain ◽  
Laser Scanning ◽  
Alveolar Epithelial Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Intracellular Replication ◽  
Alveolar Epithelial

ABSTRACT Legionella pneumophila replicates within alveolar macrophages, and possibly, alveolar epithelial cells and also within protozoa in the aquatic environment. Here we characterize an L. pneumophila mutant defective in the HtrA/DegP stress-induced protease/chaperone homologue and show that HtrA is indispensable for intracellular replication within mammalian macrophages and alveolar epithelial cells and for intrapulmonary replication in A/J mice. Importantly, amino acid substitutions of two conserved residues in the catalytic domain of (H103➤R and S212➤A) and in-frame deletions of either or both of the two conserved PDZ domains of HtrA abolish its function. Interestingly, the htrAmutant exhibits a parental-type phenotype in intracellular replication within the protozoan host Acanthamoeba polyphaga. We used a promoterless lacZ fusion to the htrApromoter to probe the phagosomal microenvironment harboringL. pneumophila within macrophages and within A. polyphaga for the exposure to stress stimuli. The data show that expression through the htrA promoter is induced by 12,000- to 20,000-fold throughout the intracellular infection of macrophages but its induction is by 120- to 500-fold within protozoa compared to in vitro expression. Data derived from confocal laser scanning microscopy reveal that in contrast to the parental strain, phagosomes harboring the htrA mutant within U937 macrophages colocalize with the late endosomal-lysosomal marker LAMP-2, similar to killed L. pneumophila. Coinfection experiments examined by confocal laser scanning microscopy show that in communal phagosomes harboring both the parental strain and the htrA mutant, replication of the mutant is not rescued, while replication of a dotAmutant control, which is normally trafficked into a phagolysosome, is rescued by the parental strain. Our data show, for the first time, that the stress response by L. pneumophila (mediated, at least in part, by HtrA) is indispensable for intracellular replication within mammalian but not protozoan cells.

Finding the comfort zone: Online-monitoring of electroactive bacteria colonising electrode surfaces with different chemical properties

2020 ◽  
Author(s):  
Hanna Frühauf ◽  
Markus Stöckl ◽  
Dirk Holtmann
Keyword(s):  
Electrochemical Impedance Spectroscopy ◽  
Impedance Spectroscopy ◽  
Confocal Laser Scanning Microscopy ◽  
Electrode Surface ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Electrochemical Impedance ◽  
Flow Cell ◽  
Laser Scanning Microscopy ◽  
Confocal Laser

<p>Mechanisms of electron transfer vary greatly within the diverse group of electroactive microorganisms and so does the need to attach to the electrode surface, e.g. by forming a biofilm.</p> <p>Electrochemical impedance spectroscopy (EIS) and confocal laser scanning microscopy (CLSM) are well established methods to monitor cell attachment to an electrode surface and have therefore been combined in a flow cell as a screening system. The flow cell, equipped with a transparent indium tin oxide working electrode (ITO WE), allows monitoring of attachment processes in real time with minimal needs for additional biofilm preparation. In preliminary experiments the flow cell was successfully used as microbial fuel cell (MFC) with a potential of +0.4 V vs. Ag/AgCl using <em>Shewanella oneidensis</em> as electroactive model organism. [1]</p> <p>Commonly, graphite-based electrode materials are used in bioelectrochemical systems due to their low costs and high conductivity. However, the hydrophobic and negatively charged surface is not yet optimal for microbial attachment. There are numerous attempts on electrode surface engineering in order to overcome this problem. In the majority of studies the biofilm analysis and evaluation of the attachment takes place at the end of the experiment, neglecting the impacts of the chemical surface properties and initial electrode conditioning during the very beginning of biofilm formation.</p> <p>To investigate initial attachment and biofilm formation in real-time, the transparent ITO-electrode is coated with polyelectrolytes differing in hydrophobicity and polarity to evaluate their effects on the initial surface colonisation by different electroactive microorganisms. Combining CLSM and EIS, both, surface coverage and electrochemical interaction of electrode-associated bacteria can be assessed.</p> <p>With this we aim to understand and ease initial steps of biofilm formation to improve efficiency of bioelectrochemical applications, e.g. with regards to start-up time.</p> <p> </p> <p>[1] Stöckl, M., Schlegel, C., Sydow, A., Holtmann, D., Ulber, R., & Mangold, K. M. (2016). Membrane separated flow cell for parallelized electrochemical impedance spectroscopy and confocal laser scanning microscopy to characterize electro-active microorganisms. <em>Electrochimica Acta</em>, 220, 444-452.</p>

scholarly journals Electrochemical and surface analytical techniques applied to microbiologically influenced corrosion investigation

2018 ◽  
Vol 36 (4) ◽  
pp. 349-363 ◽  
Author(s):  
László Trif ◽  
Abdul Shaban ◽  
Judit Telegdi
Keyword(s):  
Photoelectron Spectroscopy ◽  
Laser Scanning ◽  
Electrochemical Impedance ◽  
Electrochemical Techniques ◽  
Analytical Techniques ◽  
Laser Scanning Microscopy ◽  
Microbiologically Influenced Corrosion ◽  
Microbial Consortia ◽  
Confocal Laser ◽  
The Impact

AbstractSuitable application of techniques for detection and monitoring of microbiologically influenced corrosion (MIC) is crucial for understanding the mechanisms of the interactions and for selecting inhibition and control approaches. This paper presents a review of the application of electrochemical and surface analytical techniques in studying the MIC process of metals and their alloys. Conventional electrochemical techniques, such as corrosion potential (Ecorr), redox potential, dual-cell technique, polarization curves, electrochemical impedance spectroscopy (EIS), electrochemical noise (EN) analysis, and microelectrode techniques, are discussed, with examples of their use in various MIC studies. Electrochemical quartz crystal microbalance, which is newly used in MIC study, is also discussed. Microscopic techniques [scanning electron microscopy (SEM), environmental SEM (ESEM), atomic force microscopy (AFM), confocal laser microscopy (CLM), confocal laser scanning microscopy (CLSM), confocal Raman microscopy] and spectroscopic analytical methods [Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS)] are also highlighted. This review highlights the heterogeneous characteristics of microbial consortia and use of special techniques to study their probable effects on the metal substrata. The aim of this review is to motivate using a combination of new procedures for research and practical measurement and calculation of the impact of MIC and biofilms on metals and their alloys.

scholarly journals Temperature-Regulated Formation of Mycelial Mat-Like Biofilms by Legionella pneumophila

2006 ◽  
Vol 72 (2) ◽  
pp. 1613-1622 ◽  
Author(s):  
Zhenyu Piao ◽  
Chun Chau Sze ◽  
Oksana Barysheva ◽  
Ken-ichiro Iida ◽  
Shin-ichi Yoshida
Keyword(s):  
Legionella Pneumophila ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
C Cells ◽  
Planktonic Cells ◽  
Legionnaires Disease ◽  
Novel Strategy ◽  
Glass Surfaces

ABSTRACT Fifty strains representing 38 species of the genus Legionella were examined for biofilm formation on glass, polystyrene, and polypropylene surfaces in static cultures at 25°C, 37°C, and 42°C. Strains of Legionella pneumophila, the most common causative agent of Legionnaires' disease, were found to have the highest ability to form biofilms among the test strains. The quantity, rate of formation, and adherence stability of L. pneumophila biofilms showed considerable dependence on both temperature and surface material. Glass and polystyrene surfaces gave between two- to sevenfold-higher yields of biofilms at 37°C or 42°C than at 25°C; conversely, polypropylene surface had between 2 to 16 times higher yields at 25°C than at 37°C or 42°C. On glass surfaces, the biofilms were formed faster but attached less stably at 37°C or 42°C than at 25°C. Both scanning electron microscopy and confocal laser scanning microscopy revealed that biofilms formed at 37°C or 42°C were mycelial mat like and were composed of filamentous cells, while at 25°C, cells were rod shaped. Planktonic cells outside of biofilms or in shaken liquid cultures were rod shaped. Notably, the filamentous cells were found to be multinucleate and lacking septa, but a recA null mutant of L. pneumophila was unaffected in its temperature-regulated filamentation within biofilms. Our data also showed that filamentous cells were able to rapidly give rise to a large number of short rods in a fresh liquid culture at 37°C. The possibility of this biofilm to represent a novel strategy by L. pneumophila to compete for proliferation among the environmental microbiota is discussed.

scholarly journals Antimicrobial and Anticorrosion Activity of a Novel Composite Biocide against Mixed Bacterial Strains in Taiwanese Marine Environments

Materials ◽  
2021 ◽  
Vol 14 (20) ◽  
pp. 6156
Author(s):  
Soul-Yi Chang ◽  
Shih-Yen Huang ◽  
Yu-Ren Chu ◽  
Shun-Yi Jian ◽  
Kai-Yin Lo ◽  
...  
Keyword(s):  
Stainless Steel ◽  
Laser Scanning ◽  
Electrochemical Impedance ◽  
Laser Scanning Microscopy ◽  
304 Stainless Steel ◽  
Single Type ◽  
Confocal Laser ◽  
Subtropical Climate ◽  
Marine Environments ◽  
Bacterial Strains

Taiwan is an island with a humid subtropical climate. The relatively warm seawater results in biofouling of the surfaces of marine facilities. Biocide application is a common practice for combating and eliminating adhesive fouling. However, a single type of biocide may have limited antimicrobial effects due to the relatively high microbial diversity in marine environments. Therefore, applying a mixture of various biocides may be necessary. In this study, the antimicrobial and anticorrosion properties of a newly designed composite biocide, namely a combination of thymol and benzyldimethyldodecylammonium chloride, were investigated by applying the biocide to 304 stainless steel substrates immersed in inocula containing bacterial strains from Tamsui and Zuoying harbors. The ability of 3TB and 5TB treatments to prevent sessile cells and biofilm formation on the 304 stainless steel coupon surface was determined through scanning electron microscopy investigation. In addition, confocal laser scanning microscopy indicated that the 5TB treatment achieved a greater bactericidal effect in both the Tamsui and Zuoying inocula. Moreover, electrochemical impedance spectroscopy revealed that the diameter of the Nyquist semicircle was almost completely unaffected by Tamsui or Zuoying under the 5TB treatment. Through these assessments of antimicrobial activity and corrosion resistance, 5TB treatment was demonstrated to have superior bactericidal activity against mixed strains in both southern and northern Taiwanese marine environments.

scholarly journals Essential Role for the Legionella pneumophila Rep Helicase Homologue in Intracellular Infection of Mammalian Cells

2000 ◽  
Vol 68 (12) ◽  
pp. 6970-6978 ◽  
Author(s):  
Omar S. Harb ◽  
Yousef Abu Kwaik
Keyword(s):  
Legionella Pneumophila ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Alveolar Epithelial Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wild Type ◽  
Intracellular Replication ◽  
Simultaneous Exposure ◽  
U937 Macrophage

ABSTRACT We have previously isolated 32 mutants of Legionella pneumophila that are defective in the infection of mammalian cells but not protozoa. The mutated loci have been designated macrophage-specific infectivity (mil) loci. In this study we characterized the mil mutant GK11. This mutant was incapable of growth within U937 macrophage-like cells and WI-26 alveolar epithelial cells. This defect in intracellular replication correlated with a defect in cytopathogenicity to these cells. Sequence analysis of the GK11 locus revealed it to be highly similar torep helicase genes of other bacteria. Since helicase mutants of Escherichia coli are hypersensitive to thymine starvation, we examined the sensitivity of GK11 to thymineless death (TLD). In the absence of thymine and thymidine, mutant GK11 did not undergo TLD but was defective for in vitro growth, and the defect was partially restored when these compounds were added to the growth medium. In addition, supplementation with thymidine or thymine partially restored the ability of GK11 to grow within and kill U937 macrophage-like cells. The data suggested that the low levels of thymine or thymidine in the L. pneumophila phagosome contributed to the defect of GK11 within macrophages. Using confocal laser scanning microscopy, we determined the effect of the mutation in the Rep helicase homologue on the intracellular trafficking of GK11 within macrophages. In contrast to the wild-type strain, phagosomes harboring GK11 colocalized with several late endosomal/lysosomal markers, including LAMP-1, LAMP-2, and cathepsin D. In addition, only 50% of the GK11 phagosomes colocalized with the endoplasmic reticulum marker BiP 4 h postinfection. Colocalization of BiP with GK11 phagosomes was absent 6 h postinfection, while 90% of the wild-type phagosomes colocalized with this marker at both time points. We propose that the low level of thymine within the L. pneumophila phagosome in combination with simultaneous exposure to multiple stress stimuli results in deleterious mutations that cannot be repaired in therep helicase homologue mutant, rendering it defective in intracellular replication.

scholarly journals Study of the Corrosion Process of AZ91D Magnesium Alloy during the First Hours of Immersion in 3.5 wt.% NaCl Solution

2018 ◽  
Vol 2018 ◽  
pp. 1-20 ◽  
Author(s):  
Vanessa Mandarano Pinela ◽  
Leandro Antônio de Oliveira ◽  
Mara Cristina Lopes de Oliveira ◽  
Renato Altobelli Antunes
Keyword(s):  
Magnesium Alloy ◽  
Photoelectron Spectroscopy ◽  
Laser Scanning ◽  
Electrochemical Impedance ◽  
Corrosion Process ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Nacl Solution ◽  
X Ray ◽  
Az91d Magnesium Alloy

The AZ91D magnesium alloy was immersed in 3.5 wt.% NaCl solution at room temperature for times ranging from 1 minute up to 72 hours. The aim was to investigate the evolution of the corrosion process using confocal laser scanning microscopy (CLSM), electrochemical impedance spectroscopy, and X-ray photoelectron spectroscopy. The microstructure of the as-received alloy was initially characterized by optical microscopy and scanning electron microscopy (SEM). The crystalline phases were identified by X-ray diffractometry. The main phases were primary-α, eutectic-α, and β (Mg17Al12). Vickers microhardness markings were made on the surface of one etched sample to facilitate the identification of the same region at each different immersion time, thus enabling the observation of the corrosion process evolution. Corrosion initiates at the grain boundaries of the eutectic microconstituent and, then, propagates through primary α-grains. The β-phase was less severely attacked.

scholarly journals Development of Electrochemical Aptasensor for Lung Cancer Diagnostics in Human Blood

Sensors ◽  
2021 ◽  
Vol 21 (23) ◽  
pp. 7851
Author(s):  
Anastasiia V. Shabalina ◽  
Darya O. Sharko ◽  
Yury E. Glazyrin ◽  
Elena A. Bolshevich ◽  
Oksana V. Dubinina ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Human Blood ◽  
Laser Scanning ◽  
Electrochemical Impedance ◽  
Layer Structure ◽  
Cancer Diagnostics ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Cyclic Voltammogram ◽  
Electrochemical Aptasensor

We describe the preparation and characterization of an aptamer-based electrochemical sensor to lung cancer tumor markers in human blood. The highly reproducible aptamer sensing layer with a high density (up to 70% coverage) on the gold electrode was made. Electrochemical methods and confocal laser scanning microscopy were used to study the stability of the aptamer layer structure and binding ability. A new blocking agent, a thiolated oligonucleotide with an unrelated sequence, was applied to fill the aptamer layer’s defects. Electrochemical aptasensor signal processing was enhanced using deep learning and computer simulation of the experimental data array. It was found that the combinations (coupled and tripled) of cyclic voltammogram features allowed for distinguishing between the samples from lung cancer patients and healthy candidates with a mean accuracy of 0.73. The capacitive component from the non-Faradic electrochemical impedance spectroscopy data indicated the tumor marker’s presence in a sample. These findings allowed for the creation of highly informative aptasensors for early lung cancer diagnostics.

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