Evaluating use of ferricyanide-mediated respiration bioassays to quantify stimulatory and inhibitory effects on Escherichia coli populations

A number of recent studies have utilised ferricyanide as a respiratory mediator for microbial-based assays for determining water quality parameters such as biochemical oxygen demand (BOD) and toxicity. The majority of assays published to date obtain a result by determining the difference in ferrocyanide accumulation between a test sample and one or more control samples. However, a validation of the relationship between ferrocyanide accumulation and standard measures of cell density or viability has not yet been performed. To this end, a rapid microbially catalysed ferricyanide-mediated respiration (FM-RES) assay was compared with standard plate count (SPC) and spectrophotometer (OD₆₀₀) measurements on a growing batch culture of Escherichia coli. Good agreement was observed between all techniques, with predictable deviations noted in different phases of the growth curves. Standardised FM-RES assays showed excellent correlations with the SPC method under controlled conditions, indicating that short-term changes in microbial activity are due to a change in per-cell respiration, rather than changes in cell numbers. The FM-RES assay was then used to observe the changes in the respiration of E. coli induced by the addition of a glucose-glutamic acid (GGA) mixture, 3,5-dichlorophenol (3,5-DCP) and Ag⁺ in various combinations and concentrations. Stimulation of respiration was pronounced in the presence of GGA while both 3,5-DCP and, in particular, Ag⁺ demonstrated inhibitory respiratory effects. The results highlight the validity and suitability of ferricyanide-mediated respiration bioassays, with appropriate modification, to monitor either stimulatory effects on microbial populations, such as occurs with BOD, or inhibitory effects, such as occurs with toxicity assays.     

Toxicity assessment of chlorophenols using a mediated microbial toxicity assay

While direct toxicity assessment (DTA) is now widely recognised as a useful tool for environmental risk assessment, many existing tests fail to meet end-user needs. This article describes the significant progress made to the MICREDOX® DTA assay, developed at Lincoln Ventures Ltd, brought about by miniaturising this assay to a multi-well plate format combined with limiting current microelectrode transduction. The benefits have been reduced: preparation time, reduced assay time, lower material costs and a higher level of replication achieved. To validate the precision of the miniaturised format, the concentrations required to cause a 50% decrease in signal (EC₅₀) by an archetypal group of toxicants, the chlorophenols, were determined using two terrestrial bacterial strains, Escherichia coli K12 and Klebsiella oxytoca 13183. The assay time was then reduced by stepwise adjustment of the incubation time, from 60 down to 5 min, and the EC₅₀s reported by E. coli to each of the toxicants after 45, 30, 15 and 5 min incubations were determined. The results obtained match closely with those reported by the Activated Sludge Respiration Inhibition Test and confirm the miniaturised multi-well plate MICREDOX® DTA assay reliably reports representative EC₅₀ values for these toxicants. The previously described trends of increasing toxicity with increasing chlorine substitution and the observation that meta-substituted chlorophenols are more toxic than their ortho-substituted counterparts are also confirmed. The ability to monitor toxicity using terrestrial organisms, in volumes amenable to multi-well microtitre plates and incubations requiring only a few minutes, facilitates the rapid generation of highly reproducible, easy to operate and inexpensive DTA measurements.     

A sensitive, rapid and inexpensive way to assay pesticide toxicity based on electrochemical biosensor

We reported a rapid toxicity assay method using electrochemical biosensor for pesticides, Escherichia coli (E. coli) was taken as a model microorganism for test. In this method, we adopted ferricyanide instead of natural electron acceptor O₂, and then microbial oxidation was substantially accelerated. Toxicity assays measured the effect of toxic materials on the metabolic activity of microorganisms. The current signal of ferrocyanide produced from the metabolism was proven to be directly related to the toxicity, which could be amplified by ultramicroelectrode array (UMEA). The ratio of the electrochemical signals, recorded in the presence and absence of toxin, provided an index of inhibition. Accordingly, a direct toxicity assessment (DTA) based on chronoamperometry was proposed to detect the effect of toxic chemicals on microorganisms. 3,5-Dichlorophenol (DCP) was taken as the reference toxicant, its IC50 was estimated to be 8.0 mg/L. Three pesticides were examined using this method. IC50 values of 6.5 mg/L for Ametryn, 22 mg/L for Fenamiphos and 5.7 mg/L for Endosulfan were determined and in line with EC50 values reported in the literature. Atomic force microscopy (AFM) was also used for morphology characterization of E. coli induced by three pesticides. These results confirmed the present electrochemical method used is reliable. In addition, the electrochemical method is a sensitive, rapid and inexpensive way for toxicity assays of pesticides.     

Molecular beacons: a real-time polymerase chain reaction assay for detecting Escherichia coli from fresh produce and water

Molecular beacons (MBs) are oligonucleotide probes that fluoresce upon hybridization. The development of a real-time polymerase chain reaction (PCR) assay to detect the presence of Escherichia coli using these fluorogenic reporter molecules is reported. MBs were designed to recognize a 19-bp region of the uid A gene, coding for an enzyme β-glucuronidase. The specificity of the MB-based PCR assay was evaluated for various E. coli strains as well as bacteria species that are present in nature. The capability of the assay to detect E. coli in drinking water and produce was demonstrated. Positive detection of E. coli was demonstrated when >10¹ CFU mL⁻¹ (colony forming unit) was present in the water samples and fresh produce after 18 h of enrichment. These assays could be carried out entirely in sealed PCR tubes, enabling rapid and semiautomated detection of E. coli in food and environmental samples.     

Construction of a Tandem Repeat Peptide Sequence with Pepsin Cutting Sites to Produce Recombinant α-Melanocyte-Stimulating Hormone

The production of α-melanocyte-stimulating hormone (α-MSH), a peptide hormone composed of 13 amino acids, is attempted by recombinant expression using E. coli as the host. To achieve this aim, a synthetic gene containing eight tandem repeats of msh gene (8msh) was designed for ribosomal synthesis of 8 α-MSH. The merit of the strategy is to diminish the peptide toxicity against the host cell and to achieve a higher production yield. Pepsin cleavage sites are introduced between the peptides for enzymatic proteolysis to obtain the monomeric peptide of α-MSH. The constructed plasmid was transformed into different strains of E. coli hosts, and E. coli XL1-Blue with gene 8msh revealed the highest yield of 8 α-MSH. Although 8 α-MSH was fractionalized in the insoluble pellets after cell lysis, pepsin cleavage was able to produce soluble α-MSH peptide, as analyzed and confirmed by mass spectrometry and peptide activity assays. The production of α-MSH was quantified using HPLC with a yield of 42.9 mg/L of LB culture. This study demonstrates the feasibility of producing α-MSH using recombinant expression of tandem repeat gene. The production procedure involves minimal post-treatment and processing and can be scaled up for industrial application.     

Asymmetric reduction of a variety of ketones with a recombinant carbonyl reductase: identification of the gene encoding a versatile biocatalyst

The gene encoding a versatile biocatalyst that shows high enantioselectivity for a variety of ketones, SCR (Saccharomyces cerevisiae carbonyl reductase), has been identified, cloned, and expressed in E. coli. Recombinant E. coli co-producing SCR and GDH (glucose dehydrogenase) is an easy-to-use, synthetically useful biocatalyst, and 8 out of the 16 alcohols obtained had enantiomeric purities of >98% ee.     

Functional Characterization of Porcine NK-Lysin: A Novel Immunomodulator That Regulates Intestinal Inflammatory Response

Porcine NK-Lysine (PNKL) is a new antimicrobial peptide (AMP) identified in the small intestine. In this study, PNKL protein was obtained through heterologous expression in Escherichia coli and was estimated by SDS-PAGE at 33 kDa. The antibacterial activities of PNKL were determined using various bacterial strains and showed broad-spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria. Furthermore, E. coli K88-challenged IPEC-J2 cells were used to determine PNKL influences on inflammatory responses. Hemolytic assays showed that PNKL had no detrimental impact on cell viability. Interestingly, PNKL elevated the viability of IPEC-J2 cells exposure to E. coli K88. PNKL significantly decreased the cell apoptosis rate, and improved the distribution and abundance of tight junction protein ZO-1 in IPEC-J2 cells upon E. coli K88-challenge. Importantly, PNKL not only down regulated the expressions of inflammatory cytokines such as the IL-6 and TNF-α, but also down regulated the expressions of NF-κB, Caspase3, and Caspase9 in the E. coli K88-challenged cells. These results suggest a novel function of natural killer (NK)-lysin, and the anti-bacterial and anti-inflammatory properties of PNKL may allow it a potential substitute for conventionally used antibiotics or drugs.     

Comparative evaluation of the effects of pesticides in acute toxicity luminescence bioassays

Acute toxicity of pesticides in water was assessed singly and in mixtures using the responses of the luminescent bacterium Vibrio fischeri (BioTox™), the aquatic invertebrate Daphnia magna (Daphtoxkit™), and the MitoScan™ assay. The latter utilized fragmented mitochondria to enzymatically convert β-nicotinamide adenine dinucleotide (NADH) to its oxidized form, NAD⁺. The rate of the conversion being sensitive to type and concentration of toxicants. The pesticides tested were Carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate), Cyromazine (N-cyclopropyl-1,3,5-triazine-2,4,6-triamine), Fenamiphos (ethyl 4-methylthio-m-tolyl isopropylphosphoramidate), and Formetanate (3-dimethylaminomethyleneiminophenyl methylcarbamate). The toxicity bioassays were characterized in terms of relative sensitivity, reproducibility, range of the linear response, and the ability to reveal synergistic/antagonistic interactions among toxicants. The D. magna assay was the most sensitive and best able to detect toxic interactions of mixtures. Also, unlike the other assays used, the response of the daphnid system was linear over a 10-fold change in pesticide concentration. Relative to the BioTox™, the MitoScan™ was 2- to 11-fold more sensitive for the compounds and mixtures tested. The EC₅₀ reproducibility of all tests was within ±20% coefficient of variation; however, the lowest observable effect concentration (LOEC) were only reproducible to ±35% on average. Cyromazine was the least toxic of the pesticides tested. To test the predictive value of the concept of concentration addition, toxicities of binary and quaternary mixtures of four different pesticides were analyzed. Synergistic/antagonistic responses were most frequently observed in testing with D. magna. Synergistic/antagonistic effects were seen only in 25 and 50% of the cases with the BioTox™ and the MitoScan™ assays, respectively.     

Development and characterization of a magnetic bead-quantum dot nanoparticles based assay capable of Escherichia coli O157:H7 quantification

The development and characterization of a magnetic bead (MB)-quantum dot (QD) nanoparticles based assay capable of quantifying pathogenic bacteria is presented here. The MB-QD assay operates by having a capturing probe DNA selectively linked to the signaling probe DNA via the target genomic DNA (gDNA) during DNA hybridization. The signaling probe DNA is labeled with fluorescent QD₅₆₅ which serves as a reporter. The capturing probe DNA is conjugated simultaneously to a MB and another QD₆₅₅, which serve as a carrier and an internal standard, respectively. Successfully captured target gDNA is separated using a magnetic field and is quantified via a spectrofluorometer. The use of QDs (i.e., QD₅₆₅/QD₆₅₅) as both a fluorescence label and an internal standard increased the sensitivity of the assay. The passivation effect and the molar ratio between QD and DNA were optimized. The MB-QD assay demonstrated a detection limit of 890 zeptomolar (i.e., 10⁻²¹ mol L⁻¹) concentration for the linear single stranded DNA (ssDNA). It also demonstrated a detection limit of 87 gene copies for double stranded DNA (dsDNA) eaeA gene extracted from pure Escherichia coli (E. coli) O157:H7 culture. Its corresponding dynamic range, sensitivity, and selectivity were also presented. Finally, the bacterial gDNA of E. coli O157:H7 was used to highlight the MB-QD assay's ability to detect below the minimum infective dose (i.e., 100 organisms) of E. coli O157:H7 in water environment.     

Phenotypic and genotypic assays for the detection and identification of adhesins from pyelonephritic Escherichia coli

Four different gene clusters have been characterized so far which encode adhesins involved in the specific binding of pathogenic Escherichia coli to epithelial cells of the urinary tractus: the pap, sfa, afa and bma operons. The ability to adhere to uroepithelial cells and to interact with one or several of the specific receptors identified for each of the 4 adhesins, has been studied for 102 E. coli strains isolated from patients with pyelonephritis. These receptor-binding assays are referred to as phenotypic assays. Isolates which adhered to uroepithelial cells 68.6% produced at least 1 of the previously described adhesins. In addition, we used DNA probes to detect homologous sequences of the pap, sfa and afa operons. Genotypic assays revealed that 87.2 % of pyelonephretic E. coli contain DNA sequences related to at least 1 of the 4 operons; 78.4 %, 22.5 % and 11.8 % of the strains harboured sequences related to pap, sfa and afa operons, respectively. The afa- and sfa- adhesion determinants were commonly found associated with the presence of the pap operon (8.8 % and 18.6 %, respectively). Detection of adhesins using the genotypic approach appears to be reliable (all adhesins detected using the phenotypic approach were also detected with probes). Detection by colony hybridization was significantly higher than by phenotypic assay. Discrepancies may have been due to absence of expression of the detected operons and may have resulted from improper in vitro growth conditions, phase variation, and/or heterogeneity of the genes encoding the adhesins within a family of related sequences.     

Microbiological assay of antibiotics based on inhibition of ammonia production monitored with an ammonia electrode

A potentiometric method with an ammonia gas sensor is described for the microbiological assay of tetracycline hydrochloride, gentamicin, streptomycin, and neomycin in pharmaceutical preparations. The method is based on the inhibiting effect of antibiotics on the production of ammonia by a suspension of Escherichia coli in a nutrient solution.     

Asymmetric reduction of ketones using recombinant E. coli cells that produce a versatile carbonyl reductase with high enantioselectivity and broad substrate specificity

The gene encoding a versatile biocatalyst that shows high enantioselectivity for a variety of ketones, SCR (Saccharomyces cerevisiae carbonyl reductase), has been identified, cloned, and expressed in Escherichia coli. Two types of expression systems with high NADPH-regenerating capacities have been constructed. One is the tandem system, where the genes encoding SCR and GDH (glucose dehydrogenase) are located in the same plasmid, and the other is the two-plasmid system, where each of the SCR and GDH genes is located in separate plasmids that can coexist in one E. coli cell. Asymmetric reduction of ketones with the recombinant E. coli cells gave synthetically useful 20 alcohols, 11 of which were enantiomerically pure. The productivity of one of these products was as high as 41 g/L.     

Rapid determination of microorganisms using a flow-injection system

An automated method for the rapid determination of microorganisms using a flow-injection system is presented. Electrochemical measurement of a mediator reduced by microbial metabolism allowed the determination of fungi and bacteria in a few minutes. The lowest detection limit was 5 × 10⁶ colony-forming units (cfu) ml^?1 for Escherichia coli. Correlation between the flow-injection method and standard microbiological methods was excellent (r = 0.997, n = 4 for Beauveria bassiana; r = 0.997, n = 7 for E. coli). The flow-injection system was applied to the on-line control of an E. coli cultivation.     

Expression of Codon-Optmized Phosphoenolpyruvate Carboxylase Gene from Glaciecola sp. HTCC2999 in Escherichia coli and its Application for C4 Chemical Production

We examined the expression of the phosphoenolpyruvate carboxylase (PEPC) gene from marine bacteria in Escherichia coli using codon optimization. The codon-optimized PEPC gene was expressed in the E. coli K-12 strain W3110. SDS-PAGE analysis revealed that the codon-optimized PEPC gene was only expressed in E. coli, and measurement of enzyme activity indicated the highest PEPC activity in the E. coli SGJS112 strain that contained the codon-optimized PEPC gene. In fermentation assays, the E. coli SGJS112 produced the highest yield of oxaloacetate using glucose as the source and produced a 20-times increase in the yield of malate compared to the control. We concluded that the codon optimization enabled E. coli to express the PEPC gene derived from the Glaciecola sp. HTCC2999. Also, the expressed protein exhibited an enzymatic activity similar to that of E. coli PEPC and increased the yield of oxaloacetate and malate in an E. coli system.     

Developing new materials for paper-based diagnostics using electrospun nanofibers

The use of electrospun nanofibers as functional material in paper-based lateral flow assays (LFAs) was studied. Specific chemical features of the nanofibers were achieved by doping the base polymer, poly(lactic acid) (PLA), with poly(ethylene glycol) (PEG) and polystyrene_8K-block-poly(ethylene-ran-butylene)_25K-block-polyisoprene_10K-Brij76 (K3-Brij76) (KB). The LFAs were assembled such that the sample flowed through the nanofiber mat via capillary action. Initial investigations focused on the sustainable spinning and assembly of different polymer structures to allow the LFA format. Here, it was found that the base polymer poly(vinyl alcohol) (PVA), which was shown to function well in microfluidic biosensors, did not work in the LFA format. In contrast, PLA-based nanofibers enabled easy assembly. Three relevant features were chosen to study nanofiber-based functionalities in the LFA format: adsorption of antibodies, quantification of results, and nonspecific binding. In particular, streptavidin-conjugated sulforhodamine B (SRB)-encapsulating liposomes were captured by anti-streptavidin antibodies adsorbed on the nanofibers. Varying the functional polymer concentration within the PLA base enabled the creation of distinct capture zones. Also, a sandwich assay for the detection of Escherichia coli O157:H7 was developed using anti-E. coli antibodies as capture and reporter species with horseradish peroxidase for signal generation. A dose–response curve for E. coli with a detection limit of 1.9?×?10⁴ cells was achieved. Finally, functional polymers were used to demonstrate that nonspecific binding could be eliminated using antifouling block copolymers. The enhancement of paper-based devices using functionalized nanofibers provides the opportunity to develop a broad spectrum of sensitive and specific bioassays with significant advantages over their traditional counterparts.

Risk Assessment in Monitoring of Water Analysis of a Brazilian River

This study aimed to introduce non-parametric tests and guard bands to assess the compliance of some river water properties with Brazilian environmental regulations. Due to the heterogeneity of the measurands pH, Biochemical Oxygen Demand (BOD), manganese molar concentration, and Escherichia coli, which could be wrongly treated as outliers, as well as the non-Gaussian data, robust methods were used to calculate the measurement uncertainty. Next, based on guard bands, the compliance assessment was evaluated using this previous uncertainty information. For these four measurands, partial overlaps between their uncertainties and the specification limit could generate doubts about compliance. The non-parametric approach for calculating the uncertainty connected to the guard bands concept classified pH and BOD as “conform”, with a risk to the consumer of up to 4.0% and 4.9%, respectively; in contrast, manganese molar concentration and Escherichia coli were “not conform”, with a risk to the consumer of up to 25% and 7.4%, respectively. The methodology proposed was satisfactory because it considered the natural heterogeneity of data with non-Gaussian behavior instead of wrongly excluding outliers. In an unprecedented way, two connected statistical approaches shed light on the measurement uncertainty in compliance assessment of water analysis.     

A sensitive lateral flow biosensor for Escherichia coli O157:H7 detection based on aptamer mediated strand displacement amplification

Foodborne diseases caused by pathogens are one of the major problems in food safety. Convenient and sensitive point-of-care rapid diagnostic tests for food-borne pathogens have been a long-felt need of clinicians. Commonly used methods for pathogen detection rely on conventional culture-based tests, antibody-based assays and polymerase chain reaction (PCR)-based techniques. These methods are costly, laborious and time-consuming. Herein, we present a simple and sensitive aptamer based biosensor for rapid detection of Escherichia coli O157:H7 (E. coli O157:H7). In this assay, two different aptamers specific for the outmembrane of E. coli O157:H7 were used. One of the aptamers was used for magnetic bead enrichment, and the other was used as a signal reporter for this pathogen, which was amplified by isothermal strand displacement amplification (SDA) and further detected by a lateral flow biosensor. Only the captured aptamers on cell membrane were amplified, limitations of conventional DNA amplification based method such as false-positive can be largely reduced. The generated signals (red bands on the test zone of a lateral flow strip) can be unambiguously read out by the naked eye. As low as 10 colony forming units (CFU) of E. coli O157:H7 were detected in this study. Without DNA extraction, the reduced handling and simpler equipment requirement render this assay a simple and rapid alternative to conventional methods.     

Toxicity Assessment of Cyanide and Tetramethylene Disulfotetramine （Tetramine） Using Luminescent Bacteria Vibrio-qinghaiensis and PbO= Electrochemical Sensor

The toxicities of cyanide and tetramethylene disulfotetramine （tetramine） were evaluated by two methods of luminescent bacteria and PbO2 electrochemical sensor. Vibrio-qinghaiensis, a kind of luminescent bacteria, can produce bioluminescence and the bioluminescence was decreased with the addition of toxicants. The toxicities of cyanide and tetrarnine were expressed as 10 min-EC50 value, which was the concentration of chemical that reduces the light output by 50% after contact for 10 min. Nano PbO2 modified electrode, a rapid toxicity determination method was also described in this work. By the PbO2 modified electrode, the current responses of Escherichia coli （E. coli） were changed with the addition of toxicants. The value of 10 min-EC50 was also provided with the PbO2 electrochemical sensor. Compared with the 10 min-EC50 and detection limits （38.38 and 0.60 μg/mL for cyanide, 0.24 and 0.02 μg/mL for tetramine） with luminescent bacteria, the PbO2 sensor provided a simple and convenient method with lower 10 min-EC50 and detection limits （26.37 and 0.52 μg/mL for cyanide, 0.21 and 0.01 μg/mL for tetramine） and fast response time.     

Monitoring the production of biosynthetic human growth hormone by micro enzyme-linked immunosorbent assay

A micro enzyme-linked immunosorbent assay (e.l.i.s.a.) has been developed for monitoring the production of human growth hormone (hGH) in E. coli. The method is unusually flexible, as it is possible to modify its conditions to give a sensitive (detection limit 4 ng l^?1) or a fast (6 h) version. The assay is reproducible; the between-assay relative standard deviation is 6%. The effects of temperature, incubation time and the concentrations of protein and the most important reagents (the solid-phase antibody and the labelled antibody) are described. The utility of the method is demonstrated in experiments optimizing the conditions for production of hGH in E. coli.