Effect of Volumetric Water Content and Clover (Trifolium incarnatum) on the Survival of Escherichia coli O157:H7 in Soil

Michael J. Rothrock Jr1, Jonathan M. Frantz2, Stephanie Burnett3, Kimberly L. Cook4

1 USDA-ARS PP&SPRU, Athens, GA, 2 USDA-ARS GPRG, Toledo, OH, 3 Univ. of Maine, Orono, ME , 4 USDA-ARS AWMRU, Bowling Green, KY

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qPCR vs. Cultural “Gold” Standard

Introduction and GoalsThere has been an increased demand for minimally processed, ready-to-eat produce, including an annual 20% increase in organically-grown produce. Inherent in these production systems, especially organic systems, is the use of manures or

animal wastes as a source of nutrients for crop growth. The livestock/poultry that produce this organic fertilizer are widely considered the source of zoonotic pathogens, including E. coli O157:H7, into the “farm-to-fork” production spectrum. The application of manures/animal wastes to arable lands has resulted in human infections and soils have been shown to provide a matrix to transfer manure-borne zoonotic pathogens from the organic wastes to crops.

How do modern, molecular-based qPCR detection assays correlate to the traditional, culture-based “gold standard” quantification assays in environmental (pre-harvest) food safety studies?How do physio-chemical parameters, namely soil moisture, affect the survival of E. coli O157:H7 in soils?

How does clover, a typical cover crop used in organic vegetable production systems, affect the survival of E. coli O157:H7 within soils?

Materials and Methods• Avirulent E. coli O157:H7 strain B6914, without the stx1 and stx2 genes were used in this experiment (maintained using typical laboratory

protocols), and was added to a soil matrix (1:3 ratio of topsoil:peat-based substrate; see Table 1 for description) in 15 cm pots• 4 treatments containing 5 experimental and 3 control (no E. coli O157:H7) replicates were as follows: (1) 25% VWC unplanted, (2) 25% VWC +clover, (3) 45% unplanted, (4) 45% +clover• Prior to filling, soil substrate was mixed in a plastic bag with the required amount of nano-pure H2O to achieve the treatment set-point, controlled release fertilizer, and inocula (starting E. coli O157:H7 concentration of ~109 CFU g-1 (dry weight)). • After filling the pots, 20 seeds per pot of clover (“Cardinal Red”, Aroostook Milling, Houlton, ME) were planted by uniformly distributing the seeds over the surface of a filled pot, then adding 1 cm of soil mixture to cover the seeds. • To maintain pre-set VWC levels in real-time, the capacitance sensor automated irrigation system of Nemali and van Iersel (2006) was used, with 1 emitter per pot, and 1 volumetric water probe per treatment to trigger irrigation for that treatment (Fig. 1).

• Weekly soil samples (10 g) were aseptically collected from each replicate pot by pooling five randomly selected 2 g core samples from each pot.

• Genomic DNA was extracted using the FastDNA Spin Kit for Soils (MPBio) according to manufacturer’s specifications, and total bacteria and E. coli O157:H7 were specifically targeted and amplified via qPCR using the 16S and uidA genes, respectively (Table 2). The qPCR reactions used the PerfeCta® qPCR Supermix (Quanta Biosciences), and analyses were performed using the Realplex 2S thermal cycler (Eppendorf).

• To confirm culturable E. coli O157:H7, 45 mL of 0.1X PBS was added to 5 g of each soil sample in a sterile Whirl-Pak bag and manually homogenized for 2 mins. Serial dilutions (1:10) were made in 0.1X PBS, and 0.1 mL of each dilution was plated on LB-A agar plates. Plates were incubated overnight at 37 °C under aerobic conditions, and the E. coli O157:H7 colonies that fluoresced under UV light were counted.

• In terms of clover growth, seedling emergence was monitored daily, and 3x per week photographs were taken to document canopy coverage using Assess 2.0.

• Canopy growth rate was estimated by calculating the change in leaf canopy coverage over time using these photographs

• Data was analyzed using SAS v9.2, (proc means, anova, reg) using a 0.05 probability level for multiple comparisons among means or Bonferroni post-tests (one-way and two-way ANOVAs, respectively). Prior to statistical analyses, all bacterial and E. coli O157:H7 cell concentrations were log10-transformed based on tests of normality using the Shapiro-Wilk test statistic

Figure 1: Single pot (left) and entire treatment (right) images of irrigation set-up to maintain VWC set-points in real time. White lines are emitter lines, Black line is the volumetric water probe cable

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• qPCR-based (targeting the uidA gene) E. coli O157:H7 estimates (Figure 2: closed symbols) closely matched those obtained using the traditional culture-based direct plate counts (Fig. 2: open symbols) for the first month, but became progressively higher towards the end of the study for both the 25% and 45% treatments.

• This difference (after 1 month) between qPCR and plate count estimates was more pronounced in the 25% VWC treatments, where qPCR estimates were 2.2–2.3 logs higher by the end of the study, as compared to being only 1.2-1.6 logs higher in the 45% VWC treatments.

• Even with these divergent estimates near the end of the study, molecularly-derived concentrations of E. coli O157:H7 were significantly correlated (R2 = 0.8382 – 0.9773) to the culture-based concentrations for all four treatments, although only the 45% VWC treatments were very highly significant (a=0.001; Table 3).

This data is very similar to a recent paper looking at the correlations between the stx1virulence gene for E. coli O157:H7 and plate counts in manure-amended soil (Rogers et al, 2011)

Figure 2: Comparison of log10-transformed E. coli O157:H7 concentrations as determined by qPCR amplification of uidA gene (closed symbols) and direct plate counts (open symbols) in soil matrices maintained at (top) 25% VWC or (bottom) 45% VWC.

For E. coli O157:H7:• Significantly lower survival in the

45% VWC treatments as compared to 25% VWC (Table 4, Fig. 3A):

Survival highest in the 25% unplanted treatment (red bar) Survival lowest in the 45% planted treatment (blue bar). • Clover significantly reduced E. coli

survival, but only in the 45% VWC treatments (green vs blue bars)

No inactivation in the 25% VWC treatments (Table 5).

For Total Bacteria:• Little change throughout, but lower

survival in the 25% VWC treatments (Table 4, Fig. 3B):

Survival lowest in the 25% unplanted treatment (red bar). • No difference in treatments under

clover growth regardless of soil moisture (yellow and blue bars), or under 45% VWC whether planted or not (Table 5).

Figure 3: Effect of VWC and clover on qPCR-derived log10-transformed concentrations of (A) E. coli O157:H7, (B) total bacteria, and (C) the relative change in the E. coli O157:H7 population to the total bacteria (EC/TB). Letters above the bar indicate significantly different groups during a sampling time based on least significant difference at a 0.05 probability level for multiple comparisons among means (LSD0.05).

E. coli O157:H7/Total Bacteria (EC/TB):• The EC/TB ratio estimates followed similar patterns to the E. coli O157:H7

estimates (Fig. 3C and Fig. 3A, respectively), and in many cases the significance of most pair-wise comparisons between treatments were strengthened using the EC/TB ratio (Tables 4 and 5).

• The most noticeable difference occurred in the 25% VWC treatment (red and yellow bars).

For E. coli (Fig. 3A): +clover << -clover only @ week 3 For TB/EC (Fig. 3C): +clover << -clover @ weeks 3 - 7 • Therefore, the clover’s inhibitory effect E. coli O157:H7 survival in

the drier soil can only be determined when accounting for changes in the total bacterial community

Correlation between Clover Growth and E. coli O157:H7 Survival

ConclusionsReferences

1 Frahm, E., and U. Obst. 2003. J. Microbiol Meth 52:123-1312. Harmes, G., et. al. 2003. Environ Sci Technol 37:343-3513. Nemali, K.S. and M.W. van Iersel. 2006. Sci Hort 110:292-2974. Rogers, S.W., et. al. 2011 Appl Environ Microbiol 77:4839-4848

Contact InformationMichael J. Rothrock Jr.

ARS-PP&SPRUAthens, GA 30605

706-546-2072 (office)Michael.rothrock@ars.usda.gov (email)

Special Thanks to:James Hunt (ARS-NEPSWL)

Peggy Pinnette (ARS-NEPSWL)Nadine Nicke (ARS-NEPSWL/UMaine)

123-52

Figure 5: Correlation between projected clover leaf area (as determined by leaf canopy image analysis) and qPCR-derived log10-transformed concentrations of (A) E. coli O157:H7, (B) total bacteria, and (C) the relative change in the E. coli O157:H7 population to the total bacteria (EC/TB).

• As expected, increased soil moisture (45% VWC) resulted in a more robust leaf canopy, and significantly greater shoot and root biomass in the planted treatments (Fig. 4)

In terms of projected leaf area (Fig. 4B), minimal differences between the 25% and 45% treatments were observed within the first 3 weeks, but after that, clover growth quickly accelerated in the 45% treatment

qPCR-based estimates were highly correlated to the direct plating “gold standard”, which is important for the efficacy of using these higher-throughput, faster molecular-based assays in future soil-based, pre-harvest food safety studies on the survival of E. coli O157:H7.

Looking at E. coli O157:H7 in relation to the survival of total bacteria (EC/TB) should be further investigated as a method to assess E. coli survival since E. coli O157:H7 can be indirectly concentrated within the soils; potentially resulting in a greater human health risk.

Future work should focus on the biochemical makeup of clover exudates in agricultural soils and their specific mechanisms of inhibition, leading to the development of crop rotations/management practices to accentuate clover’s antibacterial effects not only towards E. coli O157:H7, but other important soil-associated zoonotic pathogens.

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Figure 4: Effect of VWC on clover growth. (A) Effect on clover shoot (top) and root (bottom) mass; (B) Effect on clover projected leaf area; (C) Photograph of representative 25% VWC (T5-E1) and 45% VWC (T2-E2) clover pots.

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Effect of VWC and Clover on E. coli O157:H7 Survival

• The survival of E. coli O157:H7 alone (Fig. 5A) and relative to the total bacterial community (EC/TB; Fig. 5C) were strongly correlated to clover growth

Soil moisture had no significant effect

• Very weak correlations were found between total bacteria and projected clover leaf area (Fig. 5B), regardless of moisture conditions

• While these relationships are not causative evidence of active E. coli O157:H7 suppression, they suggest a potential role in plant communities influencing the rootzone environment to suppress E. coli O157:H7 survival