Public health food-borne illness outbreaks continue even with significant improvements in pathogen detection technologies. Produce recalls have increased in frequency in recent years. Significantly, it is evident that irrigation water used in agriculture plays an important role in potentially introducing microbial contamination. Therefore, its risk management is crucial for safety assurance of the produce supply chain.

The goal of this research is the development and optimization of a novel selective medium for sensitive screening of Shiga-toxin producing E. coli and Salmonella in irrigation water. The enrichment-indicator system developed meets the demand of a method for multi-pathogen enrichment and detection in a single assay format. The method described herein allows cost effective detection of STEC and Salmonella within 24 hours. This study details development of a facile screening technique for STEC and Salmonella in irrigation water. The m-SSS medium was inclusive of a wide range of STEC and Salmonella serotypes, while exhibiting exclusivity against common background bacteria. The incorporation of a pH indicator and a monosaccharide enabled presumptive screening of common serotypes of STEC and Salmonella. Modifications on the original medium formulation (SSS medium) were made to improve sensitivity for seven major STEC and Salmonella serotypes to comply with U.S screening standards. The developed method was applied to evaluate the microbial quality of water samples from different sources. The results suggested m-SSS was able to enrich and screen for STEC and Salmonella in diverse water samples because elevated incubation temperature increased screening selectivity of m-SSS but hindered recoveries of some Salmonella in some water samples, incubation temperatures should be adjusted to optimize for targeted pathogens.

Highlights

• A selective indicator medium was developed to screen irrigation water for contamination by STEC and Salmonella sp.
• The indicator medium was generated by removal of some selective agents and addition of a pH indicator and the carbohydrate, D-trehalose.
• The indicator medium was inclusive of all the regulated STEC serotypes and a large number of Salmonella serotypes. The medium also excluded virtually all the tested non-STEC and non-Salmonella bacteria.
• The color indicator system was shown to be effective in both spiked surface water as well with naturally occurring microflora.

Fresh produce is one of the leading causes of foodborne illnesses in the United States. A wide variety of fresh produce has been implicated in Shiga toxin- producing E. coli (STEC) and Salmonella outbreaks, including romaine lettuce, sprouts, cucumbers, peppers, tomatoes, and spinach [1]. In 2018, romaine lettuce was found to be the source of two major STEC outbreaks, which caused 272 infections, 121 hospitalizations and 5 deaths [2,3]. The investigations by the FDA found that irrigation water was the source of contamination for both outbreaks [4,5]. The microbiological safety of fresh produce products remains challenging due to the open nature of fresh produce production, which makes it susceptible to contaminations from multiple sources, including soil, water, biological amendments, and wild animal activities [6]. Some pathogens can survive for an extended amount of time in the soil after irrigation, thus potentially leading to product contamination at harvest [7]. Therefore, it is crucial to adopt an effective monitoring method for the microbial quality of irrigation water. However, the detection and isolation of STEC and Salmonella presents a technical challenge necessitating time-consuming and costly laboratory procedures that often exceed the technical and financial capabilities of many small growers and reference laboratories. In this study, we developed a colorimetric screening test for STEC and Salmonella based on a highly selective enrichment medium [8]. The test was adapted to microporous filtration membrane to permit screening of irrigation water.

Tryptic Soy Broth, MI Agar, Brain Heart Infusion were obtained from Becton Dickinson (Franklin Lakes, NJ). Tryptic Soy Agar, D-Raffinose, D-Arabinose, Bromocresol Purple, Peptone from casein, D-Xylose were obtained from Sigma-Aldrich (St. Louis, MO). D-Sorbitol was obtained from Fisher Scientific (Hampton, NH). Trehalose was obtained from GoldBio (St. Louis, MO). Bile salts was obtained from Honeywell Fluka (Charlotte, NC). CHROMagarTM STEC and CHROMagarTM were obtained from CHROMagar (Paris, France). DNA purification was conducted using commercial DNA isolation from GenElute Bacterial Genomic DNA kits obtained from Millipore Sigma (St. Louis, MO). The SSS medium, commercially known as “PDX-STEC”, was prepared according to instructions from the U.S. Patent: 9518283 (31). The modified SSS medium or m-SSS medium was prepared by removal of sulfanilamide and myricetin, and addition of 0.025% bromcresol purple and 0.5% trehalose to the formulation. The modified tryptic soy broth (mTSB) was prepared by adding 0.15% (w/v) bile salts and 0.0008% (w/v) sodium novobiocin to commercial tryptic soy broth.

Bacterial strains

STEC and Salmonella strains were obtained from the Penn State University E. coli Reference Center in University Park, Pennsylvania, the Center for Disease Control and Prevention in Atlanta, Georgia, the U.S. Meat Animal Research Center (USDA Agricultural Research Services) in Clay Center, Nebraska, the American Type Culture Collection (ATCC) in Manassas, Virginia, and the University of Minnesota Veterinary Diagnostic Laboratory in Saint Paul, Minnesota. Bacterial cultures were maintained as glycerol stock at -20°C and revived in TSB at 37°C overnight before use.

Inclusivity and Exclusivity Study of SSS Medium

The inclusivity study included 50 STEC strains and 51 Salmonella serotypes. Cultures were first serially diluted into peptone water to determine the log dilution yielding plate counts of less than 10 CFU per plate (minimum concentration). Each inclusivity culture was diluted in m-SSS medium to 100 times the minimum concentration and cultured in 96-well microtiter plates (Thermo Fisher Scientific, MA) containing 200 µL m-SSS medium at 37°C for 18-24 hours. Exclusivity study included 28 isolates of closely related non- STEC and non-Salmonella strains. Exclusivity cultures were cultured in the m-SSS medium at 37°C for 18-24 hours without any dilutions. Each enrichment culture was streaked onto CHROMagarTM STEC or CHROMagarTM Salmonella, and TSA plates.

Plates were incubated at 37°C for 18-24 hours. STEC and Salmonella strains should grow in the SSS medium and give typical results (mauve-colored colonies) on CHROMagarTM STEC and CHROMagarTM Salmonella plates, respectively. The exclusivity strains should grow poorly, or not at all, in the m-SSS medium. Results were recorded for each isolate on each plate.

Carbohydrate Fermenting Ability of STEC and Salmonella

The carbohydrate-fermenting-ability of the STEC and Salmonella isolates was examined with the following five carbohydrates: D-arabinose, D-trehalose, D-sorbitol, D- raffinose, and xylose. The fermenting abilities of the strains were determined by culturing the isolates in 96 well microtiter plates containing 200 µL of bovine heart infusion (BHI) broth supplemented with bromocresol purple (0.025%, w/v) and a particular carbohydrate (1%, w/v). Positive results were identified by the color of the medium changing from purple to brown or yellow after 24 hours incubation at 37°C.

Optimization of Sensitivity of SSS Medium

Freshly prepared STEC O111, STEC O157, STEC O26, Salmonella Enteritidis, Salmonella Newport, and Salmonella Typhimurium cultures were serially diluted in sterile 0.1% peptone water. One hundred milliliters of sterile DI water samples were inoculated with 100 µL of the inoculum at different dilutions to obtain final inoculation levels ranging from 0 CFU to 105 CFU per 100 mL. After 30 min at room temperature, the samples were filtered through a sterile membrane with 0.45 µm pores to collect cells onto the membrane following the procedure previously described. The membrane was immersed and incubated in 3 mL of SSS medium at 37°C for 24 hours. The limit of detection (LOD) for each STEC and Salmonella strain was identified as the lowest inoculum level tested which resulted in a color change of the medium from purple to brown or yellow. In addition to the original SSS medium, the LOD was also determined for a modified SSS medium (m-SSS) made by removing sulfanilamide and myricetin from the formulation.

Comparison of Sensitivity and Selectivity of m-SSS with mTSB

Groundwater samples were collected from well water in Minnesota and Wisconsin. Water samples (n=16) were co-inoculated with STEC O157 and Salmonella Enteritidis, or STEC O111 and Salmonella Typhimurium, or STEC O26 and Salmonella Newport at low levels (~5 CFU per 100 mL). After 30 min at room temperature, each sample was filtered through a sterile membrane with 0.45 µm pores to collect cells onto the membrane following the procedure previously described. The membrane was immersed and incubated in 3 mL of the m-SSS medium (n=8) or mTSB (n=8) at 37°C for 24 hours. The color change of the samples in the m-SSS medium was recorded at the end of the incubation. A loopful of culture enrichment was streaked onto CHROMagarTM STEC and CHROMagarTM Salmonella bi-plate to identify positive samples. In addition, experiments were repeated with ground water samples collected from a different source with enrichment temperature at both 37°C and 42°C.

Seventeen surface water samples were collected from different sites along the St. Croix and Mississippi rivers from both Minnesota and Wisconsin, as well as lakes in Minnesota, and two ground water samples were collected from two wells in Minnesota and Wisconsin [Table 1]. One hundred milliliters of the surface water sample were filtered through a sterile membrane with 0.45 µm pores to collect cells onto the membrane following the procedure previously described. The membrane was immersed and incubated in 3 mL of m-SSS medium at 37°C or 42°C for 24 hours. A loopful of aliquot from each culture enrichment was streaked onto CHROMagarTM STEC and CHROMagarTM Salmonella bi-plate to identify positive samples. Suspect colonies were picked, and DNA purifications were conducted according to the instruction of a commercial DNA purification kit (GenElute Bacterial Genomic DNA kit). Real-time PCR analysis targeting stx1, stx2, and eae genes of STEC and the invA gene of Salmonella were performed using a Chai open PCR dual-channel instrument [Table 2]. A standard qPCR processing protocol was utilized, consisting of a 10-second denaturation at 95°C, annealing step at 60oC and an extension step at 72°C for 30 seconds. Each analysis consisted of 40 cycles. In addition, the number of generic E. coli of each water sample was determined using EPA Method 1604 [9]).

Table 1: Sampling locations of surface water.

Table 2: Sequence of DNA probe and primers used in this study [14].

To determine the LOD values of SSS and m-SSS media, triplicate experiments on different dates were performed. All data obtained were interpreted by an analysis of variance (ANOVA) using Tukey and FisherLSD tests at a significance level of 5%. Data were analyzed using OriginPro, version 9.0 (OriginPro software, OriginLab Corporation, Northampton, MA). The comparisons between probabilities of detection of pathogens using m-SSS and mTSB were conducted using Fisher’s Exact Test. A p value of larger than 0.05 was considered to indicate no significant difference in the numbers of positive test portions given by the conditions being compared. Data were analyzed using RStudio version 1.2.1335. The correlations between the generic E. coli populations and the present of STEC or Salmonella in water samples were analyzed by calculating Pearson correlation coefficient using RStudio version 1.2.1335. A Pearson correlation coefficient at 1 means a perfect positive correlation, and a Pearson correlation coefficient at -1 means a perfect negative correlation. A p value larger than 0.05 was considered to indicate no statistical significance of the reported correlation.

Both STEC and Salmonella comprise a large number of serotypes. Among all the STEC serotypes, STEC O157 is particularly important as it accounted for most of the produce outbreaks attributed to STEC [Table 3]. However, it was noted that the number of produce outbreaks attributed to STEC non-O157 serotypes has been increasing [10]. Therefore, it is important to test the inclusivity of the m-SSS medium for both STEC O157 and STEC non-O157 serotypes. Besides, inclusivity is particularly important for Salmonella as a wide range of Salmonella serotypes were implicated in foodborne illness outbreaks associated with produce [Table 4]. The inclusivity study results are depicted in Table 5 and Table 6. A significant fraction of the STEC strains used were of unknown origin because they were obtained from commercial culture collections. The proportion of STEC inclusivity strain obtained from USDA Agricultural Research Services was primarily derived from beef, veal or feedlot fecal matter. All the tested STEC strains grew well in m-SSS medium, except all the STEC O5 isolates. Additionally, one of the O91 isolates did not grow out in the SSS medium; while this O91 isolate, not surprisingly, did grow in the less selective, m-SSS medium.

Table 3: Number of STEC outbreaks attributed to produce by food category and STEC serotypes - National Outbreak Reporting System, U.S., 1998-2017 [10].

Table 4: Number of Salmonella outbreaks attributed to produce by food category and STEC serotypes - National Outbreak Reporting System, U.S., 1998-2017 [10].

Table 5: STEC inclusivity for m-SSS Medium.

Table 6: Salmonella inclusivity for m-SSS Medium.

However, it was noted that the other two STEC O91 isolates grew well in the SSS medium yet one of the O5 isolates did grow in the more selective SSS medium [Table 7]. It is not uncommon that isolates of the same bacteria serotype from difference sources exhibit heterogeneous characteristics. Mellmann et al. [11] investigated the diversity and relatedness of one hundred STEC O91 isolates. The authors were able to further divide the STEC O91 isolates into 10 different sequence types exhibiting different characteristics such as virulence. Furthermore, STEC O91 does not impose as significant public health risks as some other STEC serotypes. In fact, there was only one foodborne illness outbreak reported to CDC was attributed to STEC O91 [10]. All the regulated STEC serotypes, i.e., O157, O111, O121, O145, O45, O26, and O103, as well as several other STEC serotypes, grew well in m-SSS medium [Table 5]. All the tested Salmonella serotypes grew well in the m-SSS medium.

Table 7: STEC inclusivity for SSS Medium.

Irrigation water from different water sources contain a variety of background microflora. Although most of them are not pathogenic to humans, thus imposing little health risks, they may compete with the growth of pathogens in the medium. Therefore, it is crucial to evaluate the exclusivity of m-SSS against common background bacteria that might interfere with the enrichment of STEC and Salmonella. The exclusivity study results are depicted in Table 7. All the commensal E. coli strains grew poorly or not at all in the m-SSS medium, with the exception of two feed lot isolates. Of the 28 exclusivity bacteria tested, only Citrobacter braakii and Enterobacter gergoviae grew in the m-SSS medium. The remaining 26 strains, including E. aerogenes and E. cloacae grew poorly or not at all. Overall, the m-SSS medium exhibited excellent inclusivity and exclusivity for selective enrichment of STEC and Salmonella from irrigation water.

Results of analysis of the carbohydrate-fermenting abilities of 44 STEC strains and 25 Salmonella strains for the five carbohydrates are shown in Table 8. All STEC and Salmonella serotypes were not able to ferment arabinose within 24 hours except three STEC O26, one STEC O145, and two S. Typhimurium strains. Similarly, only STEC O26, two STEC O145 strains, and two S. Typhimurium strains were able to ferment raffinose. In general, sorbitol exhibited good fermentability by most of the tested STEC and Salmonella strains with an exception that two STEC O111 and three STEC O157 strains were not able to ferment sorbitol within 24 hours. It was unexpected that sorbitol was fermentable by one of the tested STEC O157 strains. It is believed that typical E. coli O157: H7 does not ferment sorbitol at 24 hours [12]. In fact, sorbitol has been commonly used in media formulations to differentiate E. coli O157: H7 from other fecal E. coli, such as in the MacConkey Agar with Sorbitol (SMAC). However, sorbitol-fermenting E. coli O157 strains are not uncommon. Many studies have reported isolations of sorbitol-fermenting E. coli O157 from clinical specimens [13,14]. Furthermore, sorbitol positive STEC O157:H7 mutant has also been previously reported [15]. The STEC O157 strain exhibited sorbitol fermenting ability in current study was from an unknown source. It is possible that it belongs to one of the sorbitol positive STEC O157 strains. All tested STEC and Salmonella strains were able to ferment both xylose and trehalose within 24 hours. It agrees with the previous study by Hiramatsu et al. [16]. The authors reported that all the tested STEC O26, O157, and O111 strains were able to ferment xylose and trehalose [16]. It was noted that fermentation of trehalose by some of the tested STEC and Salmonella strains were more efficient than fermentation of xylose, which resulted in a larger pH drop reflecting on a color change to a greater extent. Besides, it has been previously reported that xylose had poor fermentability among certain minor serotypes of STEC, such as O119:H4, O121:H19, O165 [HUT] [17]. Moreover, Shamanna and Sanderson [18] reported a 50% reduction of the uptake of xylose by Salmonella typhimurium LT2 when osmotic shock was applied. Therefore, trehalose appears to be a better option as the sole carbohydrate source offering advantages of applicability to a broader range of STEC serotypes and consistent efficacy for Salmonella under stress. In fact, trehalose has been shown to be an osmoregulatory solute in many bacteria, including E. coli and Salmonella, crucial for the prevention of osmotic dehydration of the cells under stressed growth environments [19,20].