title: Development of a novel cross-streaking method for isolation, confirmation, and enumeration of Salmonella from irrigation ponds.
abstract: The 2013 Produce Safety Rules in Food Safety Modernization Act (FSMA) require regular testing for generic Escherichia coli in agricultural water intended for pre-harvest contact with the edible portion of fresh produce. However, the use of fecal contamination indicators frequently does not correctly reflect distribution of foodborne pathogens such as Salmonella enterica, and ensuring food safety may require direct detection and enumeration of pathogens in agricultural settings. Herein we report the evaluation of different cost-effective methods for quantification, isolation, and confirmation of Salmonella in irrigation pond water and sediment samples. A most probably number (MPN) dual enrichment culture method was used in combination with differential and selective agars, XLT4 and CHROMagar™ Salmonella plus (CSP). The necessity for PCR confirmation was evaluated, and methods were compared by cost and performance measures (i.e., sensitivity, specificity, positive predictive value, and negative predictive value). Statistical analyses showed that using XLT4 as the initial selective agar to isolate Salmonella colonies improved recovery compared to CSP agar; however, PCR confirmation was required to avoid false positive results on either agar. Therefore, a novel cross-streaking method utilizing CHROMagar™ agar for individual colony confirmation of Salmonella presence/absence on XLT4 was developed. This method classifies the colony as positive if typical Salmonella appearance is observed on both agars. Statistical analysis showed that this method was as effective as PCR for species confirmation of pure individual strains isolated from enrichment cultures (sensitivity=0.99, specificity=1.00, relative to PCR). This method offers a cost-effective alternative to PCR that would increase the capacity and sensitivity of Salmonella evaluation.
authors.affiliation: Department of Food Science and Human Nutrition, University of Florida, Gainesville, FL 32611, USA.
authors.forename: Zhiyao
authors.initials: Z
authors.lastname: Luo
authors.affiliation: Emerging Pathogens Institute and Department of Plant Pathology, University of Florida, Gainesville, FL 32611, USA.
authors.forename: Ganyu
authors.initials: G
authors.lastname: Gu
authors.affiliation: Department of Statistics, IFAS, University of Florida, Gainesville, FL 32611, USA.
authors.forename: Mihai C
authors.initials: MC
authors.lastname: Giurcanu
authors.affiliation: Biological & Agricultural Engineering Department, University of Georgia, Tifton, GA 31793, USA.
authors.forename: Paige
authors.initials: P
authors.lastname: Adams
authors.affiliation: Biological & Agricultural Engineering Department, University of Georgia, Tifton, GA 31793, USA.
authors.forename: George
authors.initials: G
authors.lastname: Vellidis
authors.affiliation: Emerging Pathogens Institute and Department of Plant Pathology, University of Florida, Gainesville, FL 32611, USA.
authors.forename: Ariena H C
authors.initials: AH
authors.lastname: van Bruggen
authors.affiliation: Department of Food Science and Human Nutrition, University of Florida, Gainesville, FL 32611, USA. Electronic address: acw@ufl.edu.
authors.forename: Anita C
authors.initials: AC
authors.lastname: Wright
mesh_terms: D057914:Agricultural Irrigation; D001431:Bacteriological Techniques / Q000379:methods*; D003470:Culture Media; D016133:Polymerase Chain Reaction; D061005:Ponds / Q000382:microbiology*; D012475:Salmonella / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
chemical_list: D003470:Culture Media
keywords: CHROMagar™ Salmonella Plus; Cross-streaking; Irrigation ponds; PCR; Salmonella; XLT4