Understanding the costs of investigating coliform and E. coli detections during routine drinking water quality monitoring

Bacteriological failure investigations are crucial in the provision of safe, clean drinking water as part of a process of quality assurance and continual improvement. However, the financial implications of investigating coliform and Escherichia coli failures during routine water quality monitoring are poorly understood in the industry. The investigations for 737 coliform and E. coli failures across five UK water companies were analysed in this paper. The principal components of investigation costs were staff hours worked, re-samples collected, transportation, and special investigatory activities related to the sample collection location. The average investigation costs ranged from £575 for a customer tap failure to £4,775 for a water treatment works finished water failure. These costs were compared to predictions for US utilities under the Revised Total Coliform Rule. Improved understanding of the financial and staffing implications of investigating bacteriological failures can be used to budget operational expenditures and justify increased funding for preventive strategies

Macromolecular Fingerprinting of Sulfolobus Species in Biofilm: A Transcriptomic and Proteomic Approach Combined with Spectroscopic Analysis

Microorganisms in nature often live in surfaceassociated sessile communities, encased in a self-produced matrix, referred to as biofilms. Biofilms have been well studied in bacteria but in a limited way for archaea. We have recently characterized biofilm formation in three closely related hyperthermophilic crenarchaeotes: Sulfolobus acidocaldarius, S. solfataricus, and S. tokodaii. These strains form different communities ranging from simple carpet structures in S. solfataricus to high density tower-like structures in S. acidocaldarius under static condition. Here, we combine spectroscopic, proteomic, and transcriptomic analyses to describe physiological and regulatory features associated with biofilms. Spectroscopic analysis reveals that in comparison to planktonic life-style, biofilm life-style has distinctive influence on the physiology of each Sulfolobus spp. Proteomic and transcriptomic data show that biofilm-forming life-style is strain specific (eg ca. 15% of the S. acidocaldarius genes were differently expressed, S. solfataricus and S. tokodaii had ∼3.4 and ∼1%, respectively). The -omic data showed that regulated ORFs were widely distributed in basic cellular functions, including surface modifications. Several regulated genes are common to biofilm-forming cells in all three species. One of the most striking common response genes include putative Lrs14-like transcriptional regulators, indicating their possible roles as a key regulatory factor in biofilm development

Bacterial quorum sensing and cell surface electrokinetic properties

The hypothesis tested in this paper is that quorum sensing influences the microbial surface electrokinetic properties. Escherichia coli MG1655 and MG1655 LuxS- mutant (lacking quorum-sensing gene for Autoinducer synthase AI-2) were used for this study. AI-2 production (or lack of) in both strains was analyzed using the Vibrio harveyi bioassay. The levels of extracellular AI-2 with and without glucose in the growth medium were consistent with previously published work. The surface electrokinetic properties were determined for each strain of E. coli MG1655 by measuring the electrophoretic mobility using a phase amplitude light-scattering (PALS) Zeta potential analyser. The findings show that the surface charge of the cells is dependent upon the stage in the growth phase as well as the ability to participate in quorum sensing. In addition, significant differences in the electrophoretic mobility were observed between both strains of E. coli. These findings suggest that quorum sensing plays a significant role in the surface chemistry of bacteria during their growth

Strand Invasion Based Amplification (SIBA®): A Novel Isothermal DNA Amplification Technology Demonstrating High Specificity and Sensitivity for a Single Molecule of Target Analyte

<div><p>Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR) in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA). SIBA technology is resistant to non-specific amplification, is able to detect a single molecule of target analyte, and does not require target-specific probes. The technology relies on the recombinase-dependent insertion of an invasion oligonucleotide (IO) into the double-stranded target nucleic acid. The duplex regions peripheral to the IO insertion site dissociate, thereby enabling target-specific primers to bind. A polymerase then extends the primers onto the target nucleic acid leading to exponential amplification of the target. The primers are not substrates for the recombinase and are, therefore unable to extend the target template in the absence of the IO. The inclusion of 2′-O-methyl RNA to the IO ensures that it is not extendible and that it does not take part in the extension of the target template. These characteristics ensure that the technology is resistant to non-specific amplification since primer dimers or mis-priming are unable to exponentially amplify. Consequently, SIBA is highly specific and able to distinguish closely-related species with single molecule sensitivity in the absence of complex probes or sophisticated laboratory equipment. Here, we describe this technology in detail and demonstrate its use for the detection of <i>Salmonella.</i></p></div

SIBA primers are unable to amplify target DNA independently of the invasion oligonucleotide (IO).

<p>(A) Real-time monitoring of amplification using SYBR Green I, (B) melting curve analysis ((-dF (fluorescence)/dT (temperature) versus temperature), and (C) non-denaturing electrophoresis of the corresponding reaction products. Lane 1, BioRad EZ Load 20 bp Molecular Ruler (20–1000 bp); lane 2, primers + IO + template (10⁷ copies); lane 3, primers + IO + template (10⁵ copies); lane 4, primers + IO + water; lane 5, primers + template (10⁷ copies); lane 6, primers + template (10⁵ copies); lane 7, IO + template (10⁷ copies); lane 8, primers + non-homologous IO + template (10⁷ copies); lane 9, primers + IO + non-homologous template (10⁷ copies); lane 10, 200 nM primers in the absence of SIBA reaction reagents; lane 11, 200 nM IO in the absence of SIBA reaction reagents; lane 12, 200 nM primers and 200 nM IO in the absence of SIBA reagents. Lanes 10–12 served as controls for monitoring the presence of oligonucleotides in the reaction products. These were diluted in TBE buffer and run alongside the SIBA reaction products. SB-F21 and SB-R21 are the forward and reverse primers, respectively. The IO used was SB-IO. The homologous target DNA used was SB-template. nhom  =  non-homologous to the target template (SB nhom template) or non-homologous IO (SB nhom IO).</p

Sensitivity of SIBA extension to point mutations.

<p>The SIBA reaction was performed with either a fully homologous target template (SB-template) or with templates containing 1–4 base point mutation(s). The results are expressed as the delay in the threshold detection time (dt), i.e., the average Δdt =  average dt of a template containing point mutation(s) minus the average dt of the fully homologous target template (SB-template). ND denotes no detectable amplification of a template. SB-F21 and SB-R21 were the upstream and downstream primers, respectively. The invasion oligonucleotide (IO) used was SB-IO.</p><p>Sensitivity of SIBA extension to point mutations.</p

Artifactual amplification is abolished by using an invasion oligonucleotide (IO) with a 2′-O-methyl RNA modification.

<p>(A) Configuration of the IO molecules used. (B) Real-time monitoring of SIBA reactions with SYBR Green I using different IOs: (i) IO with a 2′-O-methyl RNA modification and fully homologous to the target duplex, SB-IO; (ii) IO fully homologous to the target duplex, where the 2′-O-methyl RNA modification was replaced with natural DNA nucleotides, SB-IO DNA; (iii) IO with a 2′-O-methyl RNA modification that is not homologous to the target duplex, SB-IO DIFF-METH; and (iv) IO with the 2′-O-methyl RNA modification deleted, SB-IO NON-METH. SB-F21 and SB-R21 were the forward and reverse primers, respectively. The reactions were either performed using 106 target template molecules (SB-template) or in the absence of template.</p

Mechanistic description of the SIBA reaction.

<p>All single-stranded elements are coated with gp32, except for the 2′-O-methyl RNA nucleotides. Step 1: UvsX displaces gp32 on the IO and only weakly coats the primers, since they are too short for high affinity binding. Step 2: The IO invades the complementary region of the target duplex which allows partial separation of the target duplex with the downstream end still remaining double-stranded. The out-going strand of the partially separated target duplex is stabilized by gp32. Step 3: UvsX depolymerization allows the 2′-O-methyl RNA region of the IO branch migrates into the duplex. Step 4: Both the upstream and downstream region peripheral to the IO also become short enough to dissociate. Step 5: The strand displacement polymerase is able to extend the dissociated target duplex from the primers. The forward primer displaces the IO during extension of the target template. Step 8: These events lead to the production of two copies of the target duplex. The IO is released to induce further amplification.</p