Microbial enrichment and multiplexed microfiltration for accelerated detection of Salmonella in spinach

Abstract

To attain Salmonella detection thresholds in spinach suspensions using enrichment media requires at least 24 hr. Separation and concentration of selected microorganisms via microfiltration and microfugation reduce time for sample preparation, especially when working with large volumes of vegetable suspensions. This facilitates accelerated detection of Salmonella in spinach suspensions, and may contribute to effectively monitoring this pathogen before it reaches the consumer. We report a microfiltration‐based protocol for accelerated sample preparation to concentrate and recover ≤1 colony forming unit (CFU) Salmonella/g pathogen‐free spinach. Store‐bought samples of spinach and a spinach plant subjected to two environmental conditions (temperature and light exposure) during its production were tested. The overall procedure involves extraction with buffer, a short enrichment step, prefiltration using a nylon filter, crossflow hollow fiber microfiltration, and retentate centrifugation to bring microbial cells to detection levels. Based on 1 CFU Salmonella/g frozen spinach, and a Poisson distribution statistical analyses with 99% probability, we calculated that 3 hr of incubation, when followed by microfiltration, is sufficient to reach the 2 log concentration required for Salmonella detection within 7 hr. Longer enrichment times (5 hr or more) is needed for concentrations lower than 1 CFU Salmonella/g of ready to eat spinach. The recovered microbial cells were identified and confirmed as Salmonella using both polymerase chain reaction (PCR) and plating methods. Different environmental conditions tested during production did not affect Salmonella viability; this demonstrated the broad adaptability of Salmonella and emphasized the need for methods that enable efficient monitoring of production for the presence of this pathogen.

Construction and operation of a multiplexed microfiltration device to facilitate rapid pathogen detection

“…The recovery percentages in this study (Table ) are in the expected range for BPW. In another recent work, Ku et al spiked Salmonella in spinach and also used the multiplexed device to concentrate and elute microorganisms with a back‐flushing process. Similar to our results with BPW shown here, they observed comparable recovery levels between the one‐ and four‐channel devices .…”

“…All aqueous samples to be concentrated in the multiplexed C3D may be pre‐filtered to remove large particulates, if necessary . Once prepared, the samples were poured into each sample holder and the water supply/reservoir filled with deionized water (Figure ).…”

“…When all sample holder contents were eluted, in <1 min, pumps 1 and 2 were switched “off.” Recovery vials contained ~9 ml concentrated sample. These vials were removed and immediately plated for detection; vial contents could also be centrifuged to further concentrate the sample …”

“…Estimation based on spinach experiments in Ku et al Value includes time required for short enrichment, microfiltration, and a final detection with PCR.…”

“…All aqueous samples to be concentrated in the multiplexed C3D may be pre-filtered to remove large particulates, if necessary. 3,7 Once prepared, the samples were poured into each sample holder and the water supply/reservoir filled with deionized water (Figure 1). To concentrate, the switches for pumps 1 and 2 were set to "concentrate" and "on," respectively; the permeate switch remained in the "open" position.…”