Optimized Co-extraction and Quantification of DNA From Enteric Pathogens in Surface Water Samples Near Produce Fields in California

Abstract

Pathogen contamination of surface water is a health hazard in agricultural environments primarily due to the potential for contamination of crops. Furthermore, pathogen levels in surface water are often unreported or under reported due to difficulty with culture of the bacteria. The pathogens are often present, but require resuscitation, making quantification difficult. Frequently, this leads to the use of quantitative PCR targeted to genes unique to the pathogens. However, multiple pathogen types are commonly in the same water sample, both gram + and gram –, leading to problems with DNA extraction. With Shiga toxin-producing Escherichia coli (STEC), Salmonella enterica and Listeria monocytogenes as target, a method was optimized to co-extract all three and quantify the level of each using droplet digital PCR (ddPCR). Multiplexed target genes in STEC were virulence genes, shiga toxin 2 (stx2) and hemolysin (ehx). Likewise, multiplexed targets in Listeria and Salmonella were the virulence genes listeriolysin (hly) and invasion protein A (invA). Water samples were processed using microbiological techniques for each of the pathogens and duplicate water samples were quantified by ddPCR. A significant correlation was found between culture and ddPCR results indicating detection primarily of culturable cells by ddPCR. Average virulence gene levels were 923, 23 k, 69 and 152 copies per sample for stx2, ehx, hly and invA, respectively. Additionally, stx2, ehx and inv levels were significantly correlated (P < 0.05, R = 0.34) with generic E. coli MPN levels in the duplicate samples. Indirect quantification with ddPCR will improve understanding of prevalence of the pathogens and may reduce risks associated with contaminated surface water.

Comparison of the intensity of biofilm formation by Listeria monocytogenes using classical culture-based method and digital droplet PCR

“…The hlyA gene is one of the most popular target gene for PCR amplification and has been shown to be specific for L. monocytogenes species. To date, ddPCR has been used for determination of number of foodborne pathogens (Bian et al 2015;Porcellato et al 2016;Suo et al 2010), genetically modified organisms (GMO) (Koppel et al 2015), virus HIV (Kiselinova et al 2014), soil bacteria (Kim et al 2014) and bacteria in surface waters (Cooley et al 2018). It was mainly applied in studies where a small number of bacteria in the sample was expected (Porcellato et al 2016).…”

Metagenomics as a Public Health Risk Assessment Tool in a Study of Natural Creek Sediments Influenced by Agricultural and Livestock Runoff: Potential and Limitations

“…These observations were further supported by several examples of identical MLVA types isolated from both water and sediment at the same location or downstream during periods of drought (1,15). Furthermore, the levels of pathogen in the water column and sediment are difficult to measure and are generally underestimated when using culture-based tests due to the predominance of biofilms and viable-but-not-culturable (VBNC) bacteria (19). Determining accurate pathogen levels is also problematic when using culture-independent quantitative PCR (qPCR) tests, because these tests may detect small fragments of highly degraded DNA long after the living microbe and pathogens have been inactivated (20).…”

“…PCR-based quantification method for STEC. Droplet digital PCR (ddPCR; Bio-Rad) was performed on sediment DNA according to the method described by Cooley et al (19). Each 20-l reaction mixture contained 10 l Bio-Rad Supermix for Probes, 2 l primer (0.3 M final concentration) and probe (0.2 M), up to 1 g DNA, 1.2 l MgCl 2 (1.5 mM), and 0.2 l HindIII (0.2 U/l).…”

“…Each 20-l reaction mixture contained 10 l Bio-Rad Supermix for Probes, 2 l primer (0.3 M final concentration) and probe (0.2 M), up to 1 g DNA, 1.2 l MgCl 2 (1.5 mM), and 0.2 l HindIII (0.2 U/l). Primer and probe sequences were as previously published for STEC (19). Droplets were created with Droplet Generation Oil for Probes in the QX-200 droplet generator (Bio-Rad) and amplified for 5 min at 95°C, 45 cycles at 95°C for 30 s and 60°C for 90 s, and then 5 min at 72°C, and 5 min at 98°C.…”

New strategies for the enumeration of enteric pathogens in water

“…coli and Salmonella ) pathogens from the same DNA extraction, due to an optimized DNA extraction protocol. However, the samples tested here were Moore swab samples, which contained significant levels of sediment, and may not behave the same as open water samples with regards to the fraction of live versus dead cells (Cooley et al ., ).…”