Sampling sites in Monterey County in California were selected on the basis of ease of access and have been sampled repeatedly in the last 12 years using Moore swabs (see below), deployed for 24 h at the sites in Figure 1 (Cooley et al., 2007, 2014). Sites were grouped into regions based on watershed when possible. Carr Lake is likely impacted by seepage from septic systems of Salinas. Conversely, upstream regions (Gabilan Creek, Alisal Creek and the upper portion of the Salinas River are animal-impacted as they were exposed, to a great extent, by wildlife in riparian areas, and runoff from cattle ranches (primarily cow-calf operations).

At 2-week intervals over 10-months (12/15–9/16), duplicate Moore swabs (cut cheesecloth, gathered and tethered on a single fishing line) were deployed for 24 h at the above sample sites. Swabs were placed into Whirl-Pak bags (Nasco), kept on ice during transport and one of the duplicate swabs for each location was frozen at −80°C. For the remaining swab, 500 mL of sterile water was added to each bag, followed by vigorous shaking by hand for 20 s to recover a representative sample of the sediment. From the swab eluate, 100 mL was removed for L. monocytogenes isolation, and 100 mL was removed for generic E. coli quantification. Most probable number (MPN) quantification of generic E. coli was determined by the Colilert QuantiTray 2000 method according to the manufacturer's recommendations (Idexx Laboratories). To the swab and remaining eluate in the Whirl-Pak bag, 30 mL of 10X Tryptic Soy Broth (TSB) was added, and the bag was incubated with shaking at 200 RPM at 25°C for 2 h, then 42°C for 8 h. The TSB-enriched cultures were used for STEC and Salmonella isolations. O157 STEC and non-O157 STEC were isolated by methods published previously (Cooley et al., 2013). Briefly, genomic DNA was heat-released from TSB enrichment and QPCR was performed using a multiplexed primer set to detect all shiga toxin (stx) types. The QPCR-positive TSB-enriched cultures were streaked onto CHROMagar O157 media plates (DRG International) and isolated, mauve, E. coli-like colonies were selected for a second round of QPCR using the same stx multiplex primer set. In a parallel procedure, TSB-enriched culture was subjected to Immuno Magnetic Separation (IMS) with anti-O157 antibody (Invitrogen/Dynal), and the IMS beads were spread on two types of media; modified sheep blood agar (mSBA), novabiocin and tellurite Rainbow agar (NT-RA) (Biolog) (Cooley et al., 2013). All plates were incubated at 37°C for 24 h. Suspected O157:H7 colonies were selected on the basis of colony color, and were analyzed by PCR for the presence of the O157 O-antigen synthesis (rfbE) and intimin (eae) genes (Cooley et al., 2007). Similarly, non-O157 E. coli-like colonies were selected from NT-RA (red colonies) and mSBA (blue colonies that showed hemolytic activity) and confirmed by real-time PCR using the stx multiplex primer set described above.

The same TSB enriched culture was also used for Salmonella isolation in two parallel procedures (Kalchayanand et al., 2009). Portions of the TSB-enriched culture was either subject to IMS with anti-Salmonella antibody (Dynal, Invitrogen) followed by Rappaport- Vasilliadis Soya Peptone Broth (RVS, Oxoid, Remel) or plated onto Modified Semi-solid Rappaport Vasilliadis (MSRV) medium. Colonies from both RVS and MSRV were streaked onto Xylose Desoxycholate agar (XLD, Difco, Becton Dickinson-BBL). Isolated black colonies on XLD were picked and were confirmed as Salmonella by PCR for the invA gene (Gorski et al., 2011, 2013).

Enrichment and isolation of L. monocytogenes was performed as described previously (Cooley et al., 2014). Swab aliquots were enriched with Buffered Listeria Enrichment Broth Base (BLEB, Difco) for 18 h at 30°C and subsequently subjected to IMS with anti-Listeria antibody (Dynal, Invitrogen), with two parallel methods used to detect L. monocytogenes from the beads. Aliquots of re-suspended beads were inoculated into Fraser Broth and incubated at 37°C. Isolated blackened media colonies were sub-cultured onto Brilliance Listeria Agar plate (Oxoid, Remel). A separate aliquot of the re-suspended beads was plated onto Brilliance Listeria Agar and incubated for 2 days at 37°C. Isolated blue colonies surrounded by clearing were picked and streaked onto Modified Oxford Agar (MOX). Bluish-white colonies from MOX and Brilliance were selected for detection of the hlyA gene by PCR (Cooley et al., 2014).

Examination of the published sequences of stx2 and ehx variants in STECs in GenBank revealed conserved regions for designing PCR primers and probes. Likewise, Salmonella and Listeria sequences for invA and hly were examined for conserved regions, respectively. Probes for Salmonella and Listeria were designed with an internal Nova quencher to allow for smaller probe sequence and lower base fluorescence (BioSearch). Restriction patterns were also considered to eliminate those regions where the restriction site HindIII was located within the amplicons. Likewise a unique region of the plasmid pHCred (Takara Corp) was selected as internal control (IC) within the coding region of the fluorescent protein from sea anemone Heteractis crispa. All primers and probes were examined to minimize internal hairpin and dimer formations with itself and other members of the multiplex. Primer length and/or position were also adjusted to allow optimal amplification with 60°C annealing temperature for all multiplex sets. Sequence of the selected primers and probes are listed in Table 1. Multiplex sets were constructed as STEC (stx2 and ehx), Sal/Lm (invA and hly), IC (stx2, IC). Primer and probe sequences were BLASTN at NCBI to ensure they are unique to their respective targets.

Duplicate frozen swabs, mentioned above, were thawed in 190 mL sterile water. Representative samples of the sediments in the swabs were recovered by agitation (1 min vigorous agitation by hand). Liquid was moved into sterile conical tubes and centrifuged (Sorvall) swinging bucket rotor (4,700 RPM) for 10 min at room temp. The pellet was weighed and 2–3 gm was extracted using the DNeasy PowerMax Soil Kit (MoBio Corp) following the manufacture's protocol. For extractions from samples potentially containing Listeria, different pre-treatments prior to the original PowerSoil Kit protocol were used, including lysozyme (Sigma, 10–70 mg/mL), mutanolysin (Sigma, 250 U/mL), lysostaphin (Sigma, 40 U/mL) with extended incubations at 37°C (1–3 h), beadbeating (garnet or glass beads, 1 min full power (BioSpec Mini-8), sonication (2 min at power level 4, 10 s pulses, Virtis Virsonic 550 with CV4 20 KHz converter and microprobe (3 mm). Sonication was performed in a SterilGARD III biosafety cabinet (Baker Co.). Extraction optimization and sensitivity testing was done by spiking E. coli O157 (RM1484), S. enterica (RM7323) and L. monocytogenes (RM2194) into swab pellets. Number of spiked cells was determined by plate count. Purified DNA was quantified using Nanodrop (ThermoFisher).

Droplets for Droplet Digital PCR (ddPCR, BioRad) were created following the manufacture's protocol for 20 μL reactions using 10 μL BioRad's Supermix for Probes, 2 μL primer (0.3 μM) and probe (0.2 μM) (10X mix), up to 1 μg DNA, 1 μL internal control (1 fg/μL, pHCred, Takara), 1.2 μL MgCl₂ (25 mM stock), 0.2 μL HindIII (20 U/μL). Droplets were created with Droplet Generation Oil for Probes in the QX200 droplet generator (BioRad), then transferred to a 96 well PCR plate and amplified in a thermal cycler 5 min at 95°C, 45 cycles at 95°C for 30 s and 60°C for 90 s, then 5 min at 72°C, 5 min at 98°C, 4°C overnight. Pathogen genomic DNA with their respective target gene(s) was spiked into reactions containing 100–1000 ng sediment DNA. A single copy of the target gene is expected within these genomes. Sediment samples used for these spiking experiments were previously shown to be negative for pathogen(s) using the culture method described above. Droplets were processed with the QX-200 Droplet reader and the QuantaSoft software version 1.7.4 (BioRad) using the Rare Event Detection procedure.

Confidence interval for predicted template levels was computed by QuantaSoft (BioRad) using Poisson distribution statistics. In most cases the threshold fluorescence level for positive droplet detection was automatically determined by a proprietary algorithm within QuantaSoft. Manual threshold was determined as necessary using automatic settings within duplicate or control wells as per BioRad Best Practices Guidelines. Correlations between ddPCR results (pathogen presence or absence) and culture results used the Dice Coefficient in AddinSoft XLSTAT Base version 2018. One Way ANOVA was used to determine the significance between log₁₀ transformed ddPCR results and both sample watersheds and sample seasons. Pearson Product Moment was used for correlation analysis between log₁₀ transformed ddPCR results and log₁₀ transformed 5 day prior precipitation totals and log₁₀ transformed generic E. coli MPN values. Both One Way ANOVA and Pearson Product Moment were from SigmaPlot version 11.0

Since sensitivity of pathogen detection is dependent on the amount of swab DNA added to the reactions, initial studies looked at the effect of non-target DNA levels on the ability to detect IC and the O157 strain RM1484 with stx2 as target. Non-target DNA added to these reactions came from swabs, the duplicate of which had previously been shown to contain no detectable pathogen using microbiological methods. The culture methods (<10 cells/swab for STEC) are very sensitive. Nevertheless, these swabs may contain very low levels of the targeted pathogen. Spiked reactions included 1 fg IC molecules or 10 pg of RM1484 for the stx2 target (Figure 2). At both 100 ng and 1 μg non-target swab DNA per reaction, ddPCR could detect all the spiked stx2 genes (Table 2). In contrast, only a fraction (29%) of spiked IC was detectable. Neither IC nor stx2 was detected without the corresponding DNA spike, indicating the absence of stx2 in this swab DNA and the absence of IC in swab and RM1484 DNAs. One microgram of DNA is the upper limit on the amount of DNA per 20 μl ddPCR reaction recommended by BioRad. All future ddPCR reactions will be at the 1 μg DNA level and produced in triplicate.

As a further test of the sensitivity of the ddPCR protocol, all three pathogens were spiked into sediment pellets recovered from swab samples previously shown to be negative for these pathogens. Cells were spiked at 10⁴ CFU per pellet and DNA was extracted following the basic MoBio protocol. Sensitivity demonstrated by the ddPCR reactions ranged from 0.4 (ehx) to 0.015 (hly) (Table 3). Amplification of IC was the same as above and did not show PCR inhibition as factor in these reactions (data not shown). Nevertheless, the relative insensitivity of the PCR reactions and/or poor recovery of DNA from the spiked cells was indicated. Poor DNA recovery was especially indicated for Listeria (hly) since preliminary experiments with spiked L. monocytogenes DNA did not indicate a hly reduction (data not shown). Advice from MoBio and literature review indicated several DNA extraction remedies. However, all of these remedies were adapted from extraction protocols on pure culture; very different from extraction of DNA from 10⁴ cells from sediment. Nevertheless, each of these implemented protocol changes was an improvement over the basic MoBio method (Table 3). However, only including sonication in the method brought hly detection near to the level of L. monocytogenes cells inoculated into the pellet.

The sonication/extraction method described above was subsequently used with a series of swab pellets spiked with different pathogen levels. Target genes were quantified in these DNAs to indicate the sensitivity of the ddPCR reactions (Table 4). The ddPCR method was sensitive to spiked cells to the lowest level tested, 10 cells per swab of each pathogen. The lowest fraction detectable was 5 Listeria cells (hly). All other targeted genes were detected at the spiked level (10 cells). Higher spiking levels were also detected at or close to the spiked level, with the exception of the Salmonella 1,000- and 100-cell spikes, detected at 380 and 45 inv genes, respectively. Without the spiked pathogens, the targeted genes were not detected in the swab pellets, indicating that DNA from the indigenous microflora in the pellets was not interfering with the method, even at low pathogen levels.

Using the above optimized DNA extraction technique, 36 swabs were processed and the DNA used for ddPCR with the two multiplex primer sets to detect virulence genes in STEC, Listeria and Salmonella. Presence or absence of the target virulence genes from the 36 reactions were compared with microbiological results from the duplicate swabs (Table 5). Best correlation was found with inv amplification (Dice 87.7%). Nevertheless, all virulence genes significantly correlated with their respective culture results. The range of quantification for individual genes varied considerably between samples, with the greatest template variation (0–753 k) and least correlation (Dice 75%) occurring with the gene ehx. Remarkably, there were no differences between pathogen levels and sample watersheds (Figure 1) or sample season (winter, spring, summer, or fall) (Table 6). Also, pathogen levels were not correlated with rain levels (5 day prior). However, stx2, ehx, and inv levels were significantly correlated with generic E. coli (Table 5).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.