Advances in bacterial concentration methods and their integration in portable detection platforms: A review.

Early detection and identification of microbial contaminants is crucial in many sectors, including clinical diagnostics, food quality control and environmental monitoring. Biosensors have recently gained attention among other bacterial detection technologies due to their simplicity, rapid response, selectivity, and integration/miniaturization potential in portable microfluidic platforms. However, biosensors are limited to the analysis of small sample volumes, and pre-concentration steps are necessary to reach the low sensitivity levels of few bacteria per mL required in the analysis of real clinical, industrial or environmental samples. Many platforms already exist where bacterial detection and separation/accumulation systems are integrated in a single platform, but they have not been compiled and critically analysed. This review reports on most recent advances in bacterial concentration/detection platforms with emphasis on the concentration strategy. Systems based on five concentration strategies, i.e. centrifugation, filtration, magnetic separation, electric separation or acoustophoresis, are here presented and compared in terms of processed sample volume, concentration efficiency, concentration time, ability to work with different types of samples, and integration potential, among others. The critical evaluation presented in the review is envision to facilitate the development of future platforms for fast, sensitive and in situ bacterial detection in real sample.

Integrated Photonic System for Early Warning of Cyanobacterial Blooms in Aquaponics.

Cyanobacterial blooms produce hazardous toxins, deplete oxygen, and secrete compounds that confer undesirable organoleptic properties to water. To prevent bloom appearance, the World Health Organization has established an alert level between 500 and 2000 cells·mL-1, beyond the capabilities of most optical sensors detecting the cyanobacteria fluorescent pigments. Flow cytometry, cell culturing, and microscopy may reach these detection limits, but they involve both bulky and expensive laboratory equipment or long and tedious protocols. Thus, no current technology allows fast, sensitive, and in situ detection of cyanobacteria. Here, we present a simple, user-friendly, low-cost, and portable photonic system for in situ detection of low cyanobacterial concentrations in water samples. The system integrates high-performance preconcentration elements and optical components for fluorescence measurement of specific cyanobacterial pigments, that is, phycocyanin. Phycocyanin has demonstrated to be more selective to cyanobacteria than other pigments, such as chlorophyll-a, and to present an excellent linear correlation with bacterial concentration from 102 to 104 cell·mL-1 (R2 = 0.99). Additionally, the high performance of the preconcentration system leads to detection limits below 435 cells·mL-1 after 10 min in aquaponic water samples. Due to its simplicity, compactness, and sensitivity, we envision the current technology as a powerful tool for early warning and detection of low pathogen concentrations in water samples.

Rapid Detection of Legionella pneumophila in Drinking Water, Based on Filter Immunoassay and Chronoamperometric Measurement.

Legionella is a pathogenic bacterium, ubiquitous in freshwater environments and able to colonise man-made water systems from which it can be transmitted to humans during outbreaks. The prevention of such outbreaks requires a fast, low cost, automated and often portable detection system. In this work, we present a combination of sample concentration, immunoassay detection, and measurement by chronoamperometry. A nitrocellulose microfiltration membrane is used as support for both the water sample concentration and the Legionella immunodetection. The horseradish peroxidase enzymatic label of the antibodies permits using the redox substrate 3,3',5,5'-Tetramethylbenzidine to generate current changes proportional to the bacterial concentration present in drinking water. Carbon screen-printed electrodes are employed in the chronoamperometric measurements. Our system reduces the detection time: from the 10 days required by the conventional culture-based methods, to 2-3 h, which could be crucial to avoid outbreaks. Additionally, the system shows a linear response (R2 value of 0.99), being able to detect a range of Legionella concentrations between 101 and 104 cfu·mL-1 with a detection limit (LoD) of 4 cfu·mL-1.

Impedimetric transducers based on interdigitated electrode arrays for bacterial detection - A review.

Application of the impedance spectroscopy technique to detection of bacteria has advanced considerably over the last decade. This is reflected by the large amount of publications focused on basic research and applications of impedance biosensors. Employment of modern technologies to significantly reduce dimension of impedimetric devices enable on-chip integration of interdigitated electrode arrays for low-cost and easy-to-use sensors. This review is focused on publications dealing with interdigitated electrodes as a transducer unit and different bacteria detection systems using these devices. The first part of the review deals with the impedance technique principles, paying special attention to the use of interdigitated electrodes, while the main part of this work is focused on applications ranging from bacterial growth monitoring to label-free specific bacteria detection.

New system for the detection of Legionella pneumophila in water samples.

Waterborne pathogens are a global concern for public health worldwide. Despite continuing efforts to maintain water safety, water quality is still affected by deterioration and pollution. Legionella pneumophila colonizes man-made water systems and can infect humans causing Legionnaire's disease (LD), pneumonia. The prevention of LD is a public health issue and requires specific systems to control and detect these microorganisms. Culture plate is the only technique currently approved, but requires more than 10 days to obtain results. A rapid test that inform in hours about the presence of Legionella pneumophila in water samples will improve the control of this pathogen colonization. In order to control colonization by L. pneumophila we developed a membrane filter method to capture and immunodetect this microorganism in water samples. This membrane filter is used to retain the bacteria using a nitrocellulose disc inside a home-made cartridge. Subsequently we perform the immunodetection of the bacteria retained in the nitrocellulose (blocking, antibody incubation, washings and developing). On comparing our test with the gold-standard, the most important finding is the considerably reduction in time maintaining the same detection limit. This rapid test is easily automated for L. pneumophila detection allowing a comprehensive surveillance of L. pneumophila in water facilities and reducing the variability in the analyses due to the low need for manipulation. Moreover, corrective measures may be applied the same day of the analysis. This method considerably reduces the detection time compared with the conventional, gold-standard detection culture method that requires more than 10 days, being decisive to prevent outbreaks.

Development of an integrated method of concentration and immunodetection of bacteria.

The microbial quality of water is a key aspect to avoid environmental and public health problems. The low pathogen concentration needed to produce a disease outbreak makes it essential to process large water volumes and use sensitive and specific methods such as immunoassays for its detection. In the present work, we describe the development of a device based on microfiltration membranes to integrate the concentration and the immunodetection of waterborne bacteria. A microfiltration membrane treatment protocol was designed to reduce the non-specific binding of antibodies, for which different blocking agents were tested. Thus, the proof of concept of the microbial detection system was also carried out using Escherichia coli as the bacterial pathogen model. E. coli suspensions were filtered through the membranes at 0.5 mL s-1, and the E. coli concentration measurements were made by absorbance, at 620 nm, of the resultant product of the enzymatic reaction among the horseradish peroxidase (HRP) bonded to the antibody, and the substrate 3,3',5,5'-tetramethylbenzidine (TMB). The results showed that the homemade concentration system together with the developed membrane treatment protocol is able to detect E. coli cells with a limit of detection (LoD) of about 100 CFU in 100 mL. Graphical abstract Scheme of the integrated method of concentration and immunodetection of bacteria.

Antibody test for Legionella pneumophila detection.

Legionella pneumophila is responsible for Legionnaires' disease (LD). Its detection in both environmental and clinical samples is mainly performed by culture plate method which requires up to 10days to obtain results. Nowadays, there are commercial antibodies against this bacterium, but they have not been tested against all subgroups of L. pneumophila sg 1 or serogroups 1-16 or their cross-reactions with other non-Legionella bacteria. Indeed, many of these antibodies became available when only 8 serogroups of L. pneumophila had been described. We tested 7 antibodies and found that 2 (Mab 8/5 and OBT) specifically detected all the subgroups of L. pneumophila sg 1, one without cross-reactions (Mab8/5). Moreover, the LP3IIG2 antibody detected almost all serogroups tested with lower rates of cross-reactivity, resulting in a specific sensitive antibody for the detection of L. pneumophila. LP3IIG2 presented higher rate of cross-reactivity against respiratory non-Legionella isolates, thereby contraindicating its clinical applicability.

Electrochemical performance and microbial community profiles in microbial fuel cells in relation to electron transfer mechanisms.

BACKGROUND: Microbial fuel cells (MFCs) operating with complex microbial communities have been extensively reported in the past, and are commonly used in applications such as wastewater treatment, bioremediation or in-situ powering of environmental sensors. However, our knowledge on how the composition of the microbial community and the different types of electron transfer to the anode affect the performance of these bioelectrochemical systems is far from complete. To fill this gap of knowledge, we designed a set of three MFCs with different constrains limiting direct and mediated electron transfer to the anode. RESULTS: The results obtained indicate that MFCs with a naked anode on which a biofilm was allowed unrestricted development (MFC-A) had the most diverse archaeal and bacterial community, and offered the best performance. In this MFC both, direct and mediated electron transfer, occurred simultaneously, but direct electron transfer was the predominant mechanism. Microbial fuel cells in which the anode was enclosed in a dialysis membrane and biofilm was not allowed to develop (MFC-D), had a much lower power output (about 60% lower), and a prevalence of dissolved redox species that acted as putative electron shuttles. In the anolyte of this MFC, Arcobacter and Methanosaeta were the prevalent bacteria and archaea respectively. In the third MFC, in which the anode had been covered by a cation selective nafion membrane (MFC-N), power output decreased a further 5% (95% less than MFC-A). In this MFC, conventional organic electron shuttles could not operate and the low power output obtained was presumably attributed to fermentation end-products produced by some of the organisms present in the anolyte, probably Pseudomonas or Methanosaeta. CONCLUSION: Electron transfer mechanisms have an impact on the development of different microbial communities and in turn on MFC performance. Although a stable current was achieved in all cases, direct electron transfer MFC showed the best performance concluding that biofilms are the major contributors to current production in MFCs. Characterization of the complex microbial assemblages in these systems may help us to unveil new electrogenic microorganisms and improve our understanding on their role to the functioning of MFCs.

Miniaturized metal oxide pH sensors for bacteria detection.

It is well known that the metabolic activity of some microorganisms results in changes of pH of the culture medium, a phenomenon that can be used for detection and quantification of bacteria. However, conventional glass electrodes that are commonly used for pH measurements are bulky, fragile and expensive, which hinders their application in miniaturized systems and encouraged to the search for alternatives. In this work, two types of metal oxide pH sensors have been tested to detect the metabolic activity of the bacterium Escherichia coli (E. coli). These pH sensors were produced on silicon chips with platinum metal contacts, onto which thin layers of IrOx or Ta2O5 were incorporated by two different methods (electrodeposition and e-beam sputtering, respectively). In order to facilitate measurement in small sample volumes, an Ag/AgCl pseudo-reference was also screen-printed in the chip and was assayed in parallel to an external Ag/AgCl reference electrode. As it is shown, the developed sensors generated results indistinguishable from those provided by a conventional glass pH-electrode but could be operated in significantly smaller sample volumes. After optimization of the detection conditions, the metal oxide sensors are successfully applied for detection of increasing concentrations of viable E. coli, with detection of less than 10(3)cfu mL(-1) in undiluted culture medium in just 5h.

Transient storage of electrical charge in biofilms of Shewanella oneidensis MR-1 growing in a microbial fuel cell.

Current output of microbial fuel cells (MFCs) depends on a number of engineering variables mainly related to the design of the fuel cell reactor and the materials used. In most cases the engineering of MFCs relies on the premise that for a constant biomass, current output correlates well with the metabolic activity of the cells. In this study we analyze to what extent, MFC output is also affected by the mode of operation, emphasizing how discontinuous operation can affect temporal patterns of current output. The experimental work has been carried out with Shewanella oneidensis MR-1, grown in conventional two-chamber MFCs subject to periodic interruptions of the external circuit. Our results indicate that after closure of the external circuit, current intensity shows a peak that decays back to basal values. The result suggests that the MFC has the ability to store charge during open circuit situations. Further studies using chronoamperometric analyses were carried out using isolated biofilms of Shewanella oneidensis MR-1 developed in a MFC and placed in an electrochemistry chamber in the presence of an electron donor. The results of these studies indicate that the amount of excess current over the basal level released by the biofilm after periods of circuit disconnection is proportional to the duration of the disconnection period up to a maximum of approximately 60 min. The results indicate that biofilms of Shewanella oneidensis MR-1 have the ability to store charge when oxidizing organic substrates in the absence of an external acceptor.