Detection of Escherichia coli O157:H7 in soil and water using multiplex PCR

Abstract

Aims: To evaluate the suitability of a multiplex PCR‐based assay for sensitive and rapid detection of Escherichia coli O157:H7 in soil and water. Methods and Results: Soil and water samples were spiked with E. coli O157:H7 and subjected to two stages of enrichment prior to multiplex PCR. Detection sensitivities were as high as 1 cfu ml–1 drinking water and 2 cfu g–1 soil. Starvation of E. coli O157:H7 for 35 d prior to addition to soil did not affect the ability of the assay to detect initial cell numbers as low as 10 cfu g–1 soil. Use of an 8‐h primary enrichment enabled detection of as few as 6 cfu g–1 soil, and 104 cfu g–1 soil with a 6‐h primary enrichment. When soil was inoculated with 105 cfu g–1, the PCR assay indicated persistence of E. coli O157:H7 during a 35 d incubation. However, when soil was inoculated with lower numbers of pathogen, PCR amplification signals indicated survival to be dependent on cell concentration. Conclusions: A multiplex PCR‐based assay, in combination with an enrichment strategy enabled sensitive and rapid detection of E. coli O157:H7 in soil and water. Significance and Impact of the Study: The ability to sensitively detect E.coli O157:H7 in environmental material within one working day represents a considerable advancement over alternative more time‐consuming methods for detection of this pathogen.

Detection of Viable but Nonculturable Escherichia coli O157:H7 Bacteria in Drinking Water and River Water

“…Use of the ethanol precipitation of polysaccharides and of the phenol-chloroform extraction procedures described above was required for detection of as few as 7 CFU/liter of E. coli O157:H7 bacteria in highly turbid river water. This detection limit for E. coli O157:H7 bacteria in river water samples represents an improvement of several orders of magnitude over those of previous methods for water analysis (7,8,12). Direct PCR analysis of DNA was able to detect 2 ϫ 10 5 CFU/liter of E. coli O157:H7 bacteria in ground water (8).…”

“…Direct PCR analysis of DNA was able to detect 2 ϫ 10 5 CFU/liter of E. coli O157:H7 bacteria in ground water (8). As few as 6,000 CFU/liter in surface water could be detected using 21 h of enrichment and real-time PCR (with DNA as a target) (7). A sandwich enzyme-linked immunosorbent assay was able to detect 7.5 ϫ 10 5 CFU/liter in lake water (12).…”

“…A number of methods have been developed for detection of E. coli O157:H7, but they often do not differentiate viable cells from dead ones (7,43). Specific detection of viable pathogens is important because only the viable cells have the potential to cause infections and, thus, pose a significant health risk.…”

“…Antigens may be present in both viable and dead cells, and cross-reaction with matrices may result in false-positive results (43). PCR-based methods can sensitively and specifically detect E. coli O157:H7 but cannot distinguish viable from dead cells (7). A recombinant bacteriophage-based assay (3) using a combination of fluorescence intensity and nutrient uptake analysis can differentiate viable from dead bacteria.…”

Kualitas Mikrobiologis Dan Higiene Pedagang Lawar Di Kawasan Pariwisata Kabupaten Gianyar

“…12,13,14 Faktor fasilitas sanitasi seperti kesediaan tempat sampah, mencuci alat yang digunakan setelah memasak, kebersihan alat masak, tersedianya sabun cuci, kran air untuk mencuci. Dalam penelitian ini berperan kebersihan fasilitas sanitasi yang digunakan dengan adanya kontaminasi bakteri.…”