Pathogen and Surrogate Survival in Relation to Fecal Indicator Bacteria in Freshwater Mesocosms

Abstract

The microbial quality of agricultural water for fresh produce production is determined by the presence of the fecal indicator bacterium (FIB) Escherichia coli, despite poor correlations with pathogen presence. Additional FIB, such as enterococci, have been utilized for assessing water quality. The study objective was to determine the survival times (first time to detect zero or censored) of FIB (E. coli and enterococci), surrogates (Listeria innocua, Listeria seeligeri, Salmonella enterica serovar Typhimurium, and PRD1), and pathogens (four strains each of pathogenic E. coli and Listeria monocytogenes and five Salmonella serovars) simultaneously inoculated in freshwater mesocosms exposed to diel and seasonal variations. Six separate mesocosm experiments were conducted for ≤28 days each season, with samples (sediment/water) collected each day for the first 7 days and weekly thereafter. Microorganisms survived significantly longer in sediment than in water (hazard ratio [HR] for water/sediment is 2.2; 95% confidence interval [CI], 1.79 to 2.71). Also, FIB E. coli survived significantly longer than FIB enterococcus (HR for enterococci/E. coli is 12.9 [95% CI, 8.18 to 20.37]) after adjusting for the sediment/water and lake/river effects. Differences in the area under the curve (calculated from log CFU or PFU over time) were used to assess pathogen and surrogate survival in relation to FIB. Despite sample type (sediment/water) and seasonal influences, survival rates of pathogenic Salmonella serovars were similar to those of FIB E. coli, and survival rates of L. monocytogenes and pathogenic E. coli were similar to those of FIB enterococci. Further investigation of microbial survival in water and sediment is needed to determine which surrogates are best suited to assess pathogen survival in agricultural water used in irrigation water for fresh produce. IMPORTANCE Contamination of fresh produce via agricultural water is well established. This research demonstrates that survival of fecal indicator bacteria, pathogenic microorganisms, and other bacterial and viral surrogates in freshwater differs by sample type (sediment/water) and season. Our work highlights potential risks associated with pathogen accumulation and survival in sediment and the possibility for resuspension and contamination of agricultural water used in fresh produce production. Specifically, a greater microbial persistence in sediments than in water over time was observed, along with differences in survival among microorganisms in relation to the fecal indicator bacteria E. coli and enterococci. Previous studies compared data among microbial groups in different environments. Conversely, fecal indicator bacteria, surrogates, and pathogenic microorganisms were assessed within the same water and sediment mesocosms in the present study during four seasons, better representing the agricultural aquatic environment. These data should be considered when agricultural microbial water quality criteria in fresh produce operations are being determined.

Keywords: Escherichia coli; FIB; Listeria; Listeria monocytogenes; PRD1; Salmonella; enterococci; fecal indicator bacteria; pathogens; sediment; surrogates; survival; water.

FIG 1

Survival of microorganisms over time by mesocosm type (river and lake), sample type (sediment and water), season (spring, summer, fall, and winter), and experiment 1 to 6. Mesocosms were inoculated with FIB E. coli and enterococci for each experiment, along with different pathogens and surrogates to determine microorganism survival in relation to FIB in the same water and sediment environment.

FIG 2

Kaplan-Meier curves of (A) survival probabilities among all microorganisms in sediment (n = 232) and water (n = 232) adjusted for the lake/river effect and (B) survival probabilities of FIB E. coli and enterococci (n = 48) adjusted for the sediment/water and lake/river effects. The number at risk represents the individual data points of microorganisms that could still be detected based on their presence on previous days. Fewer data points could be evaluated on subsequent days as more microorganisms became undetectable.

FIG 3

Survival of microorganisms in sediment and water in relation to FIB E. coli. The area under the curve (AUC) difference from E. coli (y axis) versus microorganism type (x axis) is shown. Microorganisms that tightly stick to zero with less variance have survival trends similar to that of FIB E. coli over time. AUC differences below zero indicate that microorganisms did not survive as long as FIB E. coli in sediment and water mesocosm samples.

FIG 4

Survival of microorganisms in sediment and water in relation to FIB enterococci. The area under the curve (AUC) difference from enterococci (y axis) versus microorganism type (x axis) is shown. Microorganisms that tightly stick to zero with less variance have survival trends similar to that of FIB enterococci over time. AUC differences above zero indicate that microorganisms survived longer than FIB enterococci in sediment and water mesocosm samples.

FIG 5

Flow diagram of water and sediment collection, sample preparation, mesocosm setup, inoculation, water and sediment sampling, and diluting and plating methods. ^, Microorganisms were cultured on respective selective medium, using 1 ml for 3M Petrifilm and either 50 or 100 μl for selective agar.