A Simple Assay to Assess Salmonella enterica Persistence in Lettuce Leaves After Low Inoculation Dose

Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host. Standard culture techniques are commonly used to quantify probiotic strains, but cell culture only measures replicating cells. In response to the stresses of processing and formulation, some fraction of the live probiotic microbes may enter a viable but non-culturable state (VBNC) in which they are dormant but metabolically active. These microbes are capable of replicating once acclimated to a more hospitable host environment. An operating definition of live probiotic bacteria that includes this range of metabolic states is needed for reliable enumeration. Alternative methods, such as fluorescent in situ hybridization (FISH), nucleic acid amplification techniques such as real-time quantitative PCR (RT-qPCR or qPCR), reverse transcriptase (RT-PCR), propidium monoazide-PCR, and cell sorting techniques such as flow cytometry (FC)/fluorescent activated cell sorting (FACS) offer the potential to enumerate both culturable and VBNC bacteria. Modern cell sorting techniques have the power to determine probiotic strain abundance and metabolic activity with rapid throughput. Techniques such as visual imaging, cell culture, and cell sorting, could be used in combination to quantify the proportion of viable microbes in various metabolic states. Consensus on an operational definition of viability and systematic efforts to validate these alternative techniques ultimately will strengthen the accuracy and reliability of probiotic strain enumeration.

Host-to-host transmission in most Salmonella serovars occurs primarily via the fecal-oral route. Salmonella enterica serovar Typhi is a human host-adapted pathogen and some S. Typhi patients become asymptomatic carriers. These individuals excrete large numbers of the bacteria in their feces and transmit the pathogen by contaminating water or food sources. The carrier state has also been described in livestock animals and is responsible for food-borne epidemics. Identification and treatment of carriers are crucial for the control of disease outbreaks. In this review, we describe recent advances in molecular profiling of human carriers and the use of animal models to identify potential host and bacterial genes involved in the establishment of the carrier state. Copyright © 2012 Elsevier Ltd. All rights reserved.

Real-time quantitative PCR (qPCR) is a widely used technique in microbial community analysis, allowing the quantification of the number of target genes in a community sample. Currently, the standard-curve (SC) method of absolute quantification is widely employed for these kinds of analysis. However, the SC method assumes that the amplification efficiency (E) is the same for both the standard and the sample target template. We analyzed 19 bacterial strains and nine environmental samples in qPCR assays, targeting the nifH and 16S rRNA genes. The E values of the qPCRs differed significantly, depending on the template. This has major implications for the quantification. If the sample and standard differ in their E values, quantification errors of up to orders of magnitude are possible. To address this problem, we propose and test the one-point calibration (OPC) method for absolute quantification. The OPC method corrects for differences in E and was derived from the ΔΔC(T) method with correction for E, which is commonly used for relative quantification in gene expression studies. The SC and OPC methods were compared by quantifying artificial template mixtures from Geobacter sulfurreducens (DSM 12127) and Nostoc commune (Culture Collection of Algae and Protozoa [CCAP] 1453/33), which differ in their E values. While the SC method deviated from the expected nifH gene copy number by 3- to 5-fold, the OPC method quantified the template mixtures with high accuracy. Moreover, analyzing environmental samples, we show that even small differences in E between the standard and the sample can cause significant differences between the copy numbers calculated by the SC and the OPC methods.