Development of a rapid pH-based biosensor to monitor and control the hygienic quality of reclaimed domestic wastewater

The re-use of treated domestic wastewater necessitates a rigorous control and rapid monitoring of the hygienic quality of the reclaimed water. For this purpose, a new pH-based biosensor was developed. The essence of the methodology of the sensor is the monitoring of the acidification due to bacterial metabolism of added glucose. To improve the sensitivity, the alkalinity of the water sample is reduced prior to monitoring the acidification. This is done by stripping CO2 at a neutral or acidic pH value. The hygienic aspect of the sensor lies in the applied temperature (37 degrees C) and the use of N2 as decarbonizing gas, thus creating conditions favorable for enteric bacteria. The developed sensor could be used onsite at an advanced stage of treatment, as an endpoint or intake quality control device. For both applications, a useful correlation was obtained between log total plate count and lag time or acidification rate, respectively. Absolute detection limits lay in the ranges of either 10(3) colony-forming units (CFU)/ml in 6 h (endpoint quality control), or less than 10(5) CFU/ml in 1 h (intake quality control).     

(-)Epigallocatechin-3-gallate inhibits gelatinase activity of some bacterial isolates from ocular infection,and limits their invasion through gelatine

The objective of this paper is to assess the gelatinase production by some ocular pathogenic bacterial strains, and evaluate the ability of (-)epigallocatechin-3-gallate (EGCg) to inhibit this gelatinase activity and thus limit bacterial invasion. The effect of EGCg on bacterial gelatinase activity was tested by classic zymography methods, while its effect on bacterial invasion was evaluated through the ability of growing bacteria to liquefy and thus penetrate a semisolid gelatine substrate. It was found that EGCg inhibits bacterial gelatinases with an IC(50) of about 0.2 mM, and limits invasion of gelatinase-positive bacteria at concentrations above 2 mM. These results show for the first time that EGCg, as well as having direct antibacterial activity, can also inhibit bacterial gelatinases, thus limiting their invasion on gelatine. Possible use of EGCg is thus suggested as an adjuvant in antibacterial chemotherapy.     

Neutrophil Extracellular Traps and Bacterial Biofilms in Middle Ear Effusion of Children with Recurrent Acute Otitis Media – A Potential Treatment Target

Background

Bacteria persist within biofilms on the middle ear mucosa of children with recurrent and chronic otitis media however the mechanisms by which these develop remain to be elucidated. Biopsies can be difficult to obtain from children and their small size limits analysis.

Methods

In this study we aimed to investigate biofilm presence in middle ear effusion (MEE) from children with recurrent acute otitis media (rAOM) and to determine if these may represent infectious reservoirs similarly to those on the mucosa. We examined this through culture, viability staining and fluorescent in situ hybridisation (FISH) to determine bacterial species present. Most MEEs had live bacteria present using viability staining (32/36) and all effusions had bacteria present using the universal FISH probe (26/26). Of these, 70% contained 2 or more otopathogenic species. Extensive DNA stranding was also present. This DNA was largely host derived, representing neutrophil extracellular traps (NETs) within which live bacteria in biofilm formations were present. When treated with the recombinant human deoxyribonuclease 1, Dornase alfa, these strands were observed to fragment.

Conclusions

Bacterial biofilms, composed of multiple live otopathogenic species can be demonstrated in the MEEs of children with rAOM and that these contain extensive DNA stranding from NETs. The NETs contribute to the viscosity of the effusion, potentially contributing to its failure to clear as well as biofilm development. Our data indicates that Dornase alfa can fragment these strands and may play a role in future chronic OM treatment.     

Classification and identification of bacteria by mass spectrometry and computational analysis

Background

In general, the definite determination of bacterial species is a tedious process and requires extensive manual labour. Novel technologies for bacterial detection and analysis can therefore help microbiologists in minimising their efforts in developing a number of microbiological applications.

Methodology

We present a robust, standardized procedure for automated bacterial analysis that is based on the detection of patterns of protein masses by MALDI mass spectrometry. We particularly applied the approach for classifying and identifying strains in species of the genus Erwinia. Many species of this genus are associated with disastrous plant diseases such as fire blight. Using our experimental procedure, we created a general bacterial mass spectra database that currently contains 2800 entries of bacteria of different genera. This database will be steadily expanded. To support users with a feasible analytical method, we developed and tested comprehensive software tools that are demonstrated herein. Furthermore, to gain additional analytical accuracy and reliability in the analysis we used genotyping of single nucleotide polymorphisms by mass spectrometry to unambiguously determine closely related strains that are difficult to distinguish by only relying on protein mass pattern detection.

Conclusions

With the method for bacterial analysis, we could identify fire blight pathogens from a variety of biological sources. The method can be used for a number of additional bacterial genera. Moreover, the mass spectrometry approach presented allows the integration of data from different biological levels such as the genome and the proteome.     

Immunomagnetic-electrochemiluminescent detection of Escherichia coli O157 and Salmonella typhimurium in foods and environmental water samples.

Hemorrhagic Escherichia coli O157:H7 strains and other virulent enteric pathogens can pose a serious health threat in tainted meats, poultry, and even drinking water. Traditional culture-based methods for assay of enteric pathogens in foods and water sources are relatively slow, and results can be ambiguous. Immunomagnetic separation (IMS) and detection methods have been investigated and appear promising for rapid bacterial assay of foods and environmental samples. In this work, a commercial sensor which combines IMS with electrochemiluminescence (ECL) detection is evaluated for detection of E. coli O157 and Salmonella typhimurium in foods and fomites. Results indicate that detection limits are in the range of 100 to 1,000 bacteria per ml in pristine buffer for E. coli O157 and S. typhimurium, respectively, or 1,000 to 2,000 bacteria per ml in food samples (depending on the sample) and that total processing and assay time is rapid (< 1 h) even in food samples. An immunologic "hook" or high-antigen-concentration prozone effect was observed above 10(4) and 10(5) bacteria per ml for E. coli O157 and S. typhimurium, respectively. IMS was accomplished in milk, juices, serum, supernatant fluids from ground beef, finely minced chicken, and fish suspensions as well as several freshwater sources and followed by ECL assay. Some samples, especially fish, gave unexpectedly high background ECL. Conversely, low ECL intensity was observed in nonfat and 2% fat milk samples, which appeared to be related to binding or entrapment of the antibody-coated magnetic beads by particulates in the milk, as revealed by microscopy. Results of this evaluation suggest the feasibility of immunomagnetic-ECL methodology for rapid, sensitive, and facile preliminary screening of various foods and fomites for the presence of virulent enteric pathogens.     

Feasibility of using real-time optical methods for detecting the presence of viable bacteria aerosols at low concentrations in clean room environments

An experimental investigation was carried out to determine the agreement between two methods of viable bacteria aerosol detection. Various amounts of Bacillus globigii (BG) spores were aerosolized in 1-s bursts into a HEPA-filtered air stream and sampled simultaneously with a fluorescence aerosol particle sensor (FLAPS) and a slit to agar biological air sampler. The slit sampler incorporated 150-mm malt extract culture plates, which were incubated at 37°C for at least 12 h before culturable BG particles were counted in terms of colony-forming units (CFU). A relationship between CFU and optically detected viable bacteria particles was determined as culturable particle concentrations decreased. Through further analytical procedures, the FLAPS showed a limit of detection (LOD) of 4.2 bacterial particle/2.5 l of sampled air or 1.7 × 10³ m⁻³. This real-time bacteria aerosol monitor could be used to detect burst contamination events during a surgical procedure. The technology may be used for developing a dose–response relationship between bacterial particle exposure and infection, a tool potentially helpful in determining patient risk.     

The specific and sensitive detection of bacterial pathogens within 4 h using bacteriophage amplification

This paper describes a novel approach, termed the 'phage amplification assay', for the rapid detection and identification of specific bacteria. The technique is based on the phage lytic cycle with plaque formation as the assay end-point. It is highly sensitive, quantitative and gives results typically within 4 h. The assay comprises four main stages : (1) phage infection of target bacterium ; (2) destruction of exogenous phage ; (3) amplification of phage within infected host and (4) plaque formation from infected host with the aid of helper bacteria. A key component of this assay is a potent virucidal agent derived from natural plant extracts, pomegranate rind extract (PRE). In combination with ferrous sulphate PRE can bring about an 11 log-cycle reduction in phage titre within 3 min. This is achieved without any injury to the infected target bacteria. Subsequently, any resulting plaques are derived only from infected target organisms. Data are presented for a range of bacterial hosts including Pseudomonas aeruginosa, Salmonella typhimurium and Staphylococcus aureus. The detection limit for Ps. aeruginosa was 40 bacteria ml⁻¹ in a time of 4 h and 600 bacteria m⁻¹ for Salm. typhimurium. Application of the principles of this technology to other bacterial genera is discussed.     

Electrochemical sensor based on Arthrobacter globiformis for cholinesterase activity determination

The sensors applied recently for determination of cholinesterase activity are mostly enzymatic amperometric sensors, in spite of their disadvantages: short life-time at ambient temperature, instability of the response, interferences, as well as passivation of the electrode surface. In the present paper a new approach for determination of cholinesterase activity was proposed, overcoming the main drawbacks of the analysis performed with amperometric enzymatic sensors. Instead of the immobilization of enzymes on a conducting electrode surface, whole cells of Arthrobacter globiformis, containing choline oxidase were fixed on a Clark type oxygen probe. Current proportional to bacteria respiration is registered as a sensor response. The application of whole cells of bacteria as a sensing element permits to achieve high stability of the response and long life-time of the sensor at ambient temperature, due to the conservation of the enzyme in its natural micro-environment inside the immobilized cells. The proposed sensor keeps its functionality more than 7 weeks stored in deionized water at ambient temperature. For the first 2 weeks the amplitude of the response decreases with only 10% and at the end of the studied 7 weeks period the response was 50% of the initial. The other advantages of the proposed sensor are: the dissolved oxygen is used as a mediator which concentration can be reliably and interferences free measured by the aim of a Clark type oxygen probe applied as a transducer; reproducible bacterial membranes can be elaborated by filtration of resuspended bacterial culture after preliminary determination of its activity; application of membranes containing lyophilized bacteria capable to be conserved infinitely long time and activated just before their application; negligible cost compared with the sensors based on immobilized enzymes. The steady-state response of the proposed bacterial sensor to choline obtained in 200 s is linear in the investigated concentration range up to 2 x 10(-4) moldm(-3), with detection limit of 8 x 10(-8) moldm(-3) and sensitivity of 4 x 10(-1) microAcm(3)mol(-1), at pH 6, temperature of 25 degrees C and stirring rate of 300 rpm. Choline is formed as a result of the catalytic hydrolysis (depending on the cholinesterase activity) of the substrate acetylcholine. Linear calibration graph for cholinesterase activity determination was obtained in the range up to 11 mUcm(-3), with a slope of 1.97 x 10(-2) microAcm(3)mU(-1), at pH 6, temperature of 25 degrees C and stirring rate of 300 rpm. The tests with reconstituted lyophilized serum with known activity used as a control sample confirm the accuracy of the proposed method. The relative error of the determination was only 2.82%.     

Visualization of grapevine root colonization by the Saharan soil isolate Saccharothrix algeriensis NRRL B-24137 using DOPE-FISH microscopy

Background and aim

There is currently a gap of knowledge regarding whether some beneficial bacteria isolated from desert soils can colonize epi- and endophytically plants of temperate regions. In this study, the early steps of the colonization process of one of these bacteria, Saccharothrix algeriensis NRRL B-24137, was studied on grapevine roots to determine if this beneficial strain can colonize a non-natural host plant. An improved method of fluorescence in situ hybridization (FISH), the double labeling of oligonucleotide probes (DOPE)-FISH technique was used to visualize the colonization behavior of such bacteria as well as to determine if the method could be used to track microbes on and inside plants.

Methods

A probe specific to Saccharothrix spp. was firstly designed. Visualization of the colonization behavior of S. algeriensis NRRL B-24137 on and inside roots of grapevine plants was then carried out with DOPE-FISH microscopy.

Results

The results showed that 10 days after inoculation, the strain could colonize the root hair zone, root elongation zone, as well as root emergence sites by establishing different forms of bacterial structures as revealed by the DOPE-FISH technique. Further observations showed that the strain could be also endophytic inside the endorhiza of grapevine plants.

Conclusions

Taking into account the natural niches of this beneficial strain, this study exemplifies that, in spite of its isolation from desert soil, the strain can establish populations as well as subpopulations on and inside grapevine plants and that the DOPE-FISH tool can allow to detect it.     

细菌群体感应信号分子的光电检测技术

群体感应（quorum sensing，QS）是一种依赖菌群密度的细菌交流系统。在探究细菌群体感应系统的调控机制中，对QS信号分子的鉴别和检测是不可或缺的环节，其对生命科学、药学等领域涉及细菌等微生物的相互作用、高效检测和作用机制解析等具有重要的参考意义。本文在总结不同类型细菌QS信号分子来源和结构的基础上，对QS信号分子的光电检测方法和技术进行了综述，重点对光电传感检测的敏感介质、传感界面、传感机制及测试效果进行探讨，同时关注了将微流控芯片分析技术应用于细菌QS信号分子原位监测的相关研究进展。     

DNase I treated DNA-PCR based detection of food pathogens immobilized by metal hydroxides

Bacterial immobilization by metal hydroxides can be used for enrichment of various bacterial strains including Gram (+) and Gram (−). The polymerase chain reaction (PCR)-based bacterial detection without enrichment culture could be implemented by concentrating bacteria from food matrix by metal hydroxides. To distinguish between viable and non-viable cells, it is often required to detect the mRNA, an indicator of viable cells. This technique, although provides accurate and reliable result, is expensive and time-consuming. Here, we report the studies on application of DNase I treatment to eliminate DNA from dead cells and subsequently detect the presence of viable pathogens by conventional PCR. It was found that treatment of immobilized cells with DNase I for 1 h at 37°C prior to DNA extraction could efficiently eliminate false positives due to the presence of non-viable cells. The technique was used to detect the presence of various pathogens in food model. The detection limits for Escherichia coli O157:H7 (384 bp), Listeria monocytogenes (482 bp), and E. coli wild type (580 bp) was 5 × 10¹ cells and that for Salmonella typhimurium (685 bp) was 5 × 10² cells in 10 ml of whole milk. An erratum to this article can be found at     

Resuscitation and quantification of stressed Escherichia coli K12 NCTC8797 in water samples

The aim of this study was to investigate the impact on numbers of using different media for the enumeration of Escherichia coli subjected to stress, and to evaluate the use of different resuscitation methods on bacterial numbers. E. coli was subjected to heat stress by exposure to 55 degrees C for 1h or to light-induced oxidative stress by exposure to artificial light for up to 8h in the presence of methylene blue. In both cases, the bacterial counts on selective media were below the limits of detection whereas on non-selective media colonies were still produced. After resuscitation in non-selective media, using a multi-well MPN resuscitation method or resuscitation on membrane filters, the bacterial counts on selective media matched those on non-selective media. Heat and light stress can affect the ability of E. coli to grow on selective media essential for the enumeration as indicator bacteria. A resuscitation method is essential for the recovery of these stressed bacteria in order to avoid underestimation of indicator bacteria numbers in water. There was no difference in resuscitation efficiency using the membrane filter and multi-well MPN methods. This study emphasises the need to use a resuscitation method if the numbers of indicator bacteria in water samples are not to be underestimated. False-negative results in the analysis of drinking water or natural bathing waters could have profound health effects.     

An acoustic method for the analysis of bacterial cells

The application of a biological electroacoustic sensor based on a lateral electric-field-excited piezoelectric resonator for the study of bacterial cells that interact with specific bacteriophages, mini-antibodies, and polyclonal antibodies was successfully demonstrated. The determined lower limit of microbialcell detection was approximately of 10³ to 10⁴ cells/mL for the duration of the assay of 10 min. The possibility of bacterial-cell detection via interaction with specific agents in the presence of extraneous microbiota was shown. The method allowed us to determine the spectrum of lytic activity of bacteriophages and the sensitivity of microbial cells to bacteriophages. The results of the study showed that application of a sensor piezoelectric lateral-field resonator is a promising technique for the detection and identification of microbial cells and determination of their phage resistance in microbiology, medicine, and veterinary medicine. Furthermore, the results of the experiments made it possible to understand the mechanisms of the processes that occur in a suspension of bacterial cells in the presence of various biological agents. The method also may provide useful information regarding biophysical mechanisms of interactions that occur in microbial populations.     

In-vitro diagnosis of single and poly microbial species targeted for diabetic foot infection using e-nose technology

Background

Effective management of patients with diabetic foot infection is a crucial concern. A delay in prescribing appropriate antimicrobial agent can lead to amputation or life threatening complications. Thus, this electronic nose (e-nose) technique will provide a diagnostic tool that will allow for rapid and accurate identification of a pathogen.

Results

This study investigates the performance of e-nose technique performing direct measurement of static headspace with algorithm and data interpretations which was validated by Headspace SPME-GC-MS, to determine the causative bacteria responsible for diabetic foot infection. The study was proposed to complement the wound swabbing method for bacterial culture and to serve as a rapid screening tool for bacteria species identification. The investigation focused on both single and poly microbial subjected to different agar media cultures. A multi-class technique was applied including statistical approaches such as Support Vector Machine (SVM), K Nearest Neighbor (KNN), Linear Discriminant Analysis (LDA) as well as neural networks called Probability Neural Network (PNN). Most of classifiers successfully identified poly and single microbial species with up to 90% accuracy.

Conclusions

The results obtained from this study showed that the e-nose was able to identify and differentiate between poly and single microbial species comparable to the conventional clinical technique. It also indicates that even though poly and single bacterial species in different agar solution emit different headspace volatiles, they can still be discriminated and identified using multivariate techniques.     

Evaluation of a microwire sensor functionalized to detect Escherichia coli bacterial cells

A functionalized microwire sensor based on dielectrophoresis (DEP) and antigen-antibody reaction was initially developed for sensitive and selective detection of E. coli O157:H7. The dynamics of gold-tungsten microwires were manipulated using an automated X-Y-Z stage and the sensing process included antibody immobilization and bacterial detection, and cell quantification. Antibodies were first immobilized on surface of the microwire to improve sensing specificity, and then coupled with DEP for capture of E. coli cells in a mixture of E. coli cells and non-conductive polystyrene beads. Afterward, fluorescein-conjugated secondary antibodies were applied to the wire for quantification of captured bacteria. Field Emission Scanning Electron Microscope (FESEM) figures and fluorescence intensities of bacteria on the wire validated the sensing mechanism. The entire immobilization and detection procedure could be completed within 30 min with simple operations. Performance of the microwire sensor was not significantly affected when conducted in orange juice. In addition, the detection limit of this sensor was about 5 bacterial cells per microwire in 1000 CFU/mL bacterial suspensions when the electric field generated at 3 MHz and 20 peak to peak voltage (V(pp)), and only targeted E. coli cells were concentrated and captured.     

Nods and 'intracellular' innate immunity

Innate immunity relies on the detection of microbial invaders by two distinct systems. One system comprises a family of membrane-bound receptors, termed the Toll-like receptors, while the other family, termed the nucleotide-binding site/leucine-rich repeat (NBS/LRR) proteins, consists of molecules that are found in the cytoplasmic compartment. These two detection systems recognize conserved molecular components of microbes including such structural motifs as lipopolysaccharide from the Gram-negative bacterial cell wall and peptidoglycan (PGN) found in the cell wall of both Gram-negative and Gram-positive bacteria. This review focuses on two members of the NBS/LRR family of proteins, Nod1 and Nod2. Recently, the microbial motifs sensed by these two molecules have been characterized. Both Nod1 and Nod2 recognize PGN, however, each requires distinct molecular motifs to attain sensing. Nod1 recognizes a naturally occurring muropeptide of PGN that presents a unique amino acid at its terminus called diaminopilemic acid (DAP). This amino acid is found mainly in the PGN of Gram-negative bacteria designating Nodl as a sensor of Gram-negative bacteria. In contrast, Nod2 can detect the minimal bioactive fragment of PGN, called muramyl dipeptide. Thus Nod2 is a general sensor of bacterial PGN. Since mutations in the gene encoding Nod2 were recently shown to be associated with the chronic inflammatory disease, Crohn's disease, these results are discussed in the context of how disrupting the interplay between host detection and bacterial aggression may lead to inflammatory diseases.     

Bacteria detection using disposable optical leaky waveguide sensors

Novel disposable absorbing material clad leaky waveguide sensor devices (LWD) have been developed for the detection of pathogenic particles such as bacteria. These chips are tailored to give the maximum extension of the evanescent field at the sensor surface in order to place the entire volume of the bacteria captured by immobilized antibodies on the chip surface within this field. This in turn increases the interaction of the light with the bacteria's bulk volume. Disposable LWD chips were fabricated at room temperature and without the use of expensive fabrication equipment. These LWDs have been characterised by detecting refractive index (RI) changes, scattering and fluorescence from bacterial spores at the sensor surface when illuminated at the coupling angle. The detection limit of Bacillus subtilis var. niger (BG) bacterial spores was 10(4) spores/ml and the illumination intensity of the spores was found to be three times greater than the illumination intensity generated using the surface plasmon resonance (SPR).     

Rapid and sensitive biosensor for Salmonella

The rapid and sensitive detection of Salmonella typhymurium based on the use of a polyclonal antibody immobilized by the Langmuir-Blodgett method on the surface of a quartz crystal acoustic wave device was demonstrated. The binding of bacteria to the surface changed the crystal resonance parameters; these were quantified by the output voltage of the sensor instrumentation. The sensor had a lower detection limit of a few hundred cells/ml, and a response time of < 100 s over the range of 10(2)-10(10) cells/ml. The sensor response was linear between bacterial concentrations of 10(2)-10(7) cells/ml, with a sensitivity of 18 mV/decade. The binding of bacteria was specific with two binding sites needed to bind a single cell. The sensors preserve approximately 75% of their sensitivity over a period of 32 days.     

Colorimetric/fluorescent bacterial sensing by agarose-embedded lipid/polydiacetylene films

Aim: Development of a new chromatic (colorimetric/fluorescence) bacterial sensor, for rapid, sensitive and versatile detection of bacterial proliferation. Methods and Results: We constructed agarose‐embedded chromatic films which produce dramatic colour changes and fluorescence transformations in response to bacterial growth. The sensing constructs comprise glass‐supported Langmuir–Schaeffer phospholipid/polydiacetylene films that undergo both blue‐red transformations and induction of intense fluorescence following interactions with bacterially secreted amphiphilic compounds that diffuse through the agarose. The agarose matrix coating the sensor film further contains growth nutrients, facilitating signal amplification through promotion of bacterial culture proliferation. The agarose layer also constitutes an effective barrier for reducing background signals not associated with the bacteria. We demonstrate the applications of the new sensor for the detection of Gram‐negative and Gram‐positive bacteria, and for screening specimens of physiological fluids (blood and urine) and foods (meat) for bacterial contaminations. Conclusions: The experiments demonstrate that the new agarose‐embedded film constructs are capable of bacterial detection through visible colour transitions and fluorescence emission recorded in conventional apparatuses. Significance and Impact of the Study: This work demonstrated a new simple chromatic platform for bacterial detection, based on the generation of easily recorded colour and fluorescence changes. The new bacterial detection scheme is highly generic and could be employed for varied practical uses, in which, rapid reporting on bacterial presence is required.     

Rapid biosensor for detection of antibiotic-selective growth of Escherichia coli

A rapid biosensor for the detection of bacterial growth was developed using micromechanical oscillators coated in common nutritive layers. The change in resonance frequency as a function of the increasing mass on a cantilever array forms the basis of the detection scheme. The calculated mass sensitivity according to the mechanical properties of the cantilever sensor is approximately 50 pg/Hz; this mass corresponds to an approximate sensitivity of approximately 100 Escherichia coli cells. The sensor is able to detect active growth of E. coli cells within 1 h. The starting number of E. coli cells initially attached to the sensor cantilever was, on average, approximately 1,000 cells. Furthermore, this method allows the detection of selective growth of E. coli within only 2 h by adding antibiotics to the nutritive layers. The growth of E. coli was confirmed by scanning electron microscopy. This new sensing method for the detection of selective bacterial growth allows future applications in, e.g., rapid antibiotic susceptibility testing.