Democratizing water monitoring: Implementation of a community-based qPCR monitoring program for recreational water hazards.

Recreational water monitoring can be challenging due to the highly variable nature of pathogens and indicator concentrations, the myriad of potential biological hazards to measure for, and numerous access points, both official and unofficial, that are used for recreation. The aim of this study was to develop, deploy, and assess the effectiveness of a quantitative polymerase chain reaction (qPCR) community-based monitoring (CBM) program for the assessment of bacterial and parasitic hazards in recreational water. This study developed methodologies for performing qPCR 'in the field,' then engaged with water management and monitoring groups and tested the method in a real-world implementation study to evaluate the accuracy of CBM using qPCR both quantitatively and qualitatively. This study found high reproducibility between qPCR results performed by non-expert field users and expert laboratory results, suggesting that qPCR as a methodology could be amenable to a CBM program

Dual Expression Profile of Type VI Secretion System Immunity Genes Protects Pandemic <i>Vibrio cholerae</i>

<div><p>The <i>Vibrio cholerae</i> type VI secretion system (T6SS) assembles as a molecular syringe that injects toxic protein effectors into both eukaryotic and prokaryotic cells. We previously reported that the <i>V. cholerae</i> O37 serogroup strain V52 maintains a constitutively active T6SS to kill other Gram-negative bacteria while being immune to attack by kin bacteria. The pandemic O1 El Tor <i>V. cholerae</i> strain C6706 is T6SS-silent under laboratory conditions as it does not produce T6SS structural components and effectors, and fails to kill <i>Escherichia coli</i> prey. Yet, C6706 exhibits full resistance when approached by T6SS-active V52. These findings suggested that an active T6SS is not required for immunity against T6SS-mediated virulence. Here, we describe a dual expression profile of the T6SS immunity protein-encoding genes <i>tsiV1</i>, <i>tsiV2</i>, and <i>tsiV3</i> that provides pandemic <i>V. cholerae</i> strains with T6SS immunity and allows T6SS-silent strains to maintain immunity against attacks by T6SS-active bacterial neighbors. The dual expression profile allows transcription of the three genes encoding immunity proteins independently of other T6SS proteins encoded within the same operon. One of these immunity proteins, TsiV2, protects against the T6SS effector VasX which is encoded immediately upstream of <i>tsiV2</i>. VasX is a secreted, lipid-binding protein that we previously characterized with respect to T6SS-mediated virulence towards the social amoeba <i>Dictyostelium discoideum</i>. Our data suggest the presence of an internal promoter in the open reading frame of <i>vasX</i> that drives expression of the downstream gene <i>tsiV2</i>. Furthermore, VasX is shown to act in conjunction with VasW, an accessory protein to VasX, to compromise the inner membrane of prokaryotic target cells. The dual regulatory profile of the T6SS immunity protein-encoding genes <i>tsiV1</i>, <i>tsiV2</i>, and <i>tsiV3</i> permits <i>V. cholerae</i> to tightly control T6SS gene expression while maintaining immunity to T6SS activity.</p></div

Bacterial strains.

<p>Bacterial strains.</p

VasX compromises the integrity of the inner membrane in target cells.

<p>(A) VasX dissipates the target cell's membrane potential. C6706Δ<i>tsiV2</i> harboring the plasmids indicated on the <i>x</i>-axis were analyzed using the BacLight Membrane Potential Kit and flow cytometry. The red/green fluorescence ratio was calculated for each condition. Carbonyl cyanide <i>m</i>-chlorophenyl hydrazone (CCCP) is a chemical that uncouples the proton gradient and was used as a positive control for dissipation of membrane potential in this experiment. Arabinose was included in all samples (except in the sample noted as “not induced”) to drive expression from the P_BAD promoter. These data are representative of three independent experiments. Error bars indicate standard deviation. *** = p<0.001, ** = p<0.005 relative to SecP::vasX (induced, -CCCP). p-values were calculated using the Student's one-tailed, paired t-test. (B) Cells producing periplasmic VasX are permeable to propidium iodide (PI). The strain indicated at the top of each histogram (living or ethanol-killed) was incubated in the presence of PI and analyzed by flow cytometry. P2 represents cells not permeable to PI and P3 represents cells permeable to PI. The percentage of cells represented in P2 and P3 populations is indicated. These data represent three independent experiments.</p

VasW plays an accessory role in VasX-mediated bacterial killing.

<p>(A) VasW is required for V52 to kill <i>Vibrio parahaemolyticus</i> RIMD. Survival of rifampicin-resistant RIMD was determined by measuring CFU following exposure to the indicated rifampicin-sensitive predator listed on the <i>x</i>-axis. Arabinose was included where indicated (“induced”) to drive expression from the P_BAD promoter. These data represent three independent experiments. Error bars indicate the standard deviation. (B) VasW is required for V52 to kill C6706Δ<i>tsiV2</i>. Survival of rifampicin-resistant C6706Δ<i>tsiV2</i> was determined by measuring CFU following exposure to the indicated rifampicin-sensitive predator listed on the <i>x</i>-axis. Arabinose was included where indicated (“induced”) to drive expression from the P_BAD promoter. These data represent three independent experiments. Error bars indicate the standard deviation. (C) V52Δ<i>vgrG-3</i> with deletions in <i>vasW</i> or <i>vasX</i> does not secrete Hcp. Bacterial pellet and supernatant samples from the strains indicated at the top of the blot were subjected to western blotting with α-Hcp and α-DnaK (loading and lysis control) antibodies. (D) V52Δ<i>vasW</i> does not secrete VasX. Bacterial pellet and supernatant samples from the strains indicated at the top of the blot were subjected to western blotting with α-VasX and α-DnaK (loading and lysis control) antibodies.</p

<i>VasX</i> contains a promoter to drive expression of <i>tsiV2</i> in several <i>V. cholerae</i> strain backgrounds.

<p>(A) V52 becomes susceptible to killing following deletion of <i>vasH</i> and <i>vasX</i>. Rifampicin-sensitive V52 derivatives (predator) were mixed with rifampicin-resistant V52, V52Δ<i>vasH</i>, V52Δ<i>vasX</i>, V52Δ<i>vasH</i>Δ<i>vasX</i>, and <i>E. coli</i> MG1655 (prey). Surviving prey were enumerated by selection on LB agar containing rifampicin and the results were plotted. Data are representative of three independent experiments. Error bars indicate the standard deviation. (B) The internal <i>tsiV2</i> promoter is recognized in several strains. <i>V. cholerae</i> strains indicated on the <i>x</i>-axis were transformed with pAH6-vasX(2208-3258) or plasmid control. Transformed strains were subjected to β-galactosidase assays and the Miller units were calculated and plotted. Data represent two independent experiments performed in triplicate; error bars indicate the standard deviation. *** = p<0.001, ** = p<0.005, * = p<0.01 relative to the empty vector control. n.s; not significant. p-values were calculated based on the Student's one-tailed, paired t-test.</p

Dual expression profile of the immunity protein-encoding gene <i>tsiV2</i>.

<p>(A) Schematic representation of <i>vasX</i> fragments cloned upstream of <i>lacZ</i> in the plasmid pAH6. (B, C, D) A promoter exists within <i>vasX</i>. β-galactosidase assays were performed using the strains indicated at the top of the graph. Fragments of <i>vasX</i>, or the <i>hcp-2</i> promoter present in pAH6 are indicated on the <i>x</i>-axis. Data are representative of two independent experiments performed in triplicate and the error bars indicate the standard deviation. *** = p<0.001, ** = p<0.005, * = p<0.01 relative to the empty vector control. p-values were calculated based on the Student's one-tailed, paired t-test.</p