Evaluation of an Improved Culture Method for Isolating Salmonella and Comparisons with Two Nonisolation Assays

Abstract

A newly developed plating medium, xylose-lysine-tergitol 4 (XLT4) agar, used for isolating non-typhi Salmonella, was examined for selectivity and isolation efficiency. The growth characteristics of numerous gram-negative bacteria were observed on XLT4 plates. Also, using 97 naturally-contaminated layer chicken farm drag-swab samples, the XLT4 agar significantly improved the Salmonella isolation rate compared to the other enteric plating media evaluated. In addition, a modified Salmonella culture isolation method incorporating XLT4, two novobiocin-supplemented plating media, and delayed secondary enrichment (DSE) of all negative primary sample broths, was compared to a traditional culture isolation method with unmodified plating media streaked from the same primary sample broths. A total of 155 naturally-contaminated broiler chicken farm drag-swab samples were used for this evaluation. The traditional isolation method detected 60 Salmonella-positive samples. The modified isolation method detected 13 additional Salmonella-positive samples for a total of 73. The single most significant improvement in the modified isolation method was the incorporation of XLT4 plates. The use of DSE did not significantly improve the Salmonella recovery rate. Only one additional negative sample broth became positive following DSE. This may reflect the increased isolation efficiency on the XLT4 plates from the primary enrichment broth cultures. Finally, the traditional and modified isolation methods were compared with two non isolation assays: an enzyme-linked immunosorbent assay (ELISA) and a colorimetric DNA hybridization assay (DNAHA). The same 155 broiler chicken farm drag-swab samples were used for this comparison. When each isolation method was compared with the ELISA and DNAHA techniques, the modified isolation method showed better agreement with the two non isolation assays. The specificity of both the ELISA and DNAHA increased and the sensitivity of each decreased when the results were matched with the findings from the modified isolation method. This was due to a dramatic decrease in the number of ELISA and DNAHA false-positive results and an increase in ELISA and DNAHA false-negative results because more positive samples were detected by the modified isolation method. The data from these studies indicate the crucial need for a dependable overall culture method, including highly selective isolation media, to validate judgements about the effectiveness of ELISA and DNAHA non isolation assays. When examining naturally-contaminated specimens, the true measurement of ELISA and DNAHA is largely dependent on the use of a culture isolation method with a similar or better detection threshold to verify to results of the non isolation assays.