Development of a biosensor protein bullet as a fluorescent method for fast detection of Escherichia coli in drinking water

Drinking water can be exposed to different biological contaminants from the source, through the pipelines, until reaching the final consumer or industry. Some of these are pathogenic bacteria and viruses which may cause important gastrointestinal or systemic diseases. The microbiological quality of drinking water relies mainly in monitoring three indicator bacteria of faecal origin, Escherichia coli, Enterococcus faecalis and Clostridium perfringens, which serve as early sentinels of potential health hazards for the population. Here we describe the analysis of three chimeric fluorescent protein bullets as biosensor candidates for fast detection of E. coli in drinking water. Two of the chimeric proteins (based on GFP-hadrurin and GFP-pb5 chimera proteins) failed with respect to specificity and/or sensitivity, but the GFP-colS4 chimera protein was able to carry out specific detection of E. coli in drinking water samples in a procedure encompassing about 8 min for final result and this biosensor protein was able to detect in a linear way between 20 and 103 CFU of this bacterium. Below 20 CFU, the system cannot differentiate presence or absence of the target bacterium. The fluorescence in this biosensor system is provided by the GFP subunit of the chimeric protein, which, in the case of the better performing sensor bullet, GFP-colS4 chimera, is covalently bound to a flexible peptide bridge and to a bacteriocin binding specifically to E. coli cells. Once bound to the target bacteria, the excitation step with 395 nm LED light causes emission of fluorescence from the GFP domain, which is amplified in a photomultiplier tube, and finally this signal is converted into an output voltage which can be associated with a CFU value and these data distributed along mobile phone networks, for example. This method, and the portable fluorimeter which has been developed for it, may contribute to reduce the analysis time for detecting E. coli presence in drinking water

Confocal microscopy images of <i>E</i>. <i>coli</i> (A–G, a–g), <i>Salmonella enterica</i> (H, h) and <i>Enterobacter cloacae</i> (I, i) fluorescent labeling tests.

<p>Each group of 4 images correspond to a different chimeric biosensor fluorescent protein. Images from “A” to “I” correspond to confocal microscopy, showing the fluorescent labelled cells, and images from “a” to “i” are the same confocal images but fused with an optical image generating a single photo (merged snapshot for testing that fluorescent spots actually correspond to labelled cells, and not to background fluorescence). The symbol “✽” means that the sample has been filtered in order to get rid of unbound chimera protein. A-B, images of <i>E</i>. <i>coli</i> labelled with GFP-hadrurin fluorescent chimera protein, where labeling is lost after a filtering step. C-D, images of <i>E</i>. <i>coli</i> labelled with GFP-pb5 fluorescent chimera protein, where labeling is also lost after a filtering step. Only the fluorescent biosensor chimera protein GFP-colS4 (E,F) is able to maintain its strong binding to the <i>E</i>. <i>coli</i> surface after the filtration step. Also, the other two bacterial species (negative controls) are not labelled at all (H, I). Images G and g are negative controls for <i>E</i>. <i>coli</i>, where no fluorescent biosensor chimera protein has been added, as a method to test that this bacterial cells do not show autofluorescence under these conditions.</p

Functional blocks of the measurement system at the portable <i>E</i>. <i>coli</i> Analyzer device.

<p>Functional blocks of the measurement system at the portable <i>E</i>. <i>coli</i> Analyzer device.</p

Development of a biosensor protein bullet as a fluorescent method for fast detection of <i>Escherichia coli</i> in drinking water - Fig 2

<p>A: Hypothetical spatial structure of the fluorescent biosensor chimera protein GFP-hadrurin. B: Diagram showing the attachement process of the fluorescent biosensor chimera protein to the membrane of <i>E</i>. <i>coli</i>. PMT: photomultiplier (part of the <i>E</i>. <i>coli</i> Analyzer device in charge of detecting and amplifying the fluorescence signal produced by the GFP domain of each biosensor chimera protein, after receiving the 395 nm excitation light from the LED source).</p

MUSCLE multiple sequence alignment of the OmpW proteins from <i>E</i>. <i>coli</i>, <i>S</i>. <i>enterica</i> var. <i>arizonae</i> and <i>E</i>. <i>cloacae</i>, highlighting in yellow the region encompasing the the Asp¹¹⁶, His¹¹⁷ and Glu¹²⁰ amino acids of OmpW (in blue letters: D, H and E), which are freely exposed to the extracellular space and act as binding motifs for colicin S4 bacteriocin (as well as for GFP-colS4 chimera protein in this biosensor).

<p>As it is shown, the other two enterobacteria species do not have the conserved motif DH—E as in <i>E</i>. <i>coli</i>. Accesion numbers: SAI89619 (<i>E</i>. <i>cloacae</i>), BAA14788 (<i>E</i>. <i>coli</i>), OSE54138 (<i>S</i>. <i>enterica</i>).</p