Quantitative detection of Salmonella enterica and the specific interaction with Lactuca sativa

Salmonella is among the most commonly known bacterial pathogens to cause human illness. Often Salmonellosis is associated with the consumption of contaminated foods like meat, eggs or egg products. However, during the last decades an increase of outbreaks is recognized to be caused by human pathogenic bacteria in association with fresh produce. The use of manure for production of vegetables, e.g. lettuce, contributes significantly to the risk of contamination of fresh produce. Enteric pathogens like Salmonella associated with manure can come in close contact with plants like lettuce, and a better understanding of the interaction between lettuce and Salmonella serovars during cultivation is necessary to be able to take preventive actions to reduce the risk for human health.This thesis describes the development of detection methods of Salmonellaenterica and Escherichia coli O157:H7 for routine diagnostic screening in the food production chain. Next to that, it describes the physiological and molecular interaction between Salmonella serovars and lettuce. Background information concerning Salmonella serovars in association with lettuce, like history of produce-associated outbreaks, approaches to detect the pathogen in food samples, pathogenesis, plant responses and the molecular interaction between plants and human pathogens, is discussed in Chapter one.Chapter two concerns the comparison of different molecular methods to detect S. enterica ( invA -gene) or E.coli O157:H7 ( stx -1, stx- 2 and eae -gene) with respect to sensitivity, precision and accuracy. Two basic methods were selected, both based on real-time Taqman PCR, a method that generates fluorescence upon specific DNA amplification. The increase in fluorescence during PCR is directly correlated to the amount of target DNA present after each amplification cycle. The detection and quantification methods were improved by the addition of a general internal amplification control (IAC), viz. comprised of DNA coding forgreenfluorescent protein ( gfp ), that allowed the identification of false negative results. The IAC provided insight in amplification efficiency and enabled a more accurate quantification. Implementation of the IAC did not affect the precision of the methods, although the sensitivity was reduced 10-fold. At least 1 pg of target DNA (equal to 200 CFU) was detected and quantified with high precision and accuracy. Qualitative detection was feasible even down to 10fgof target DNA (equal to 2 CFU) per reaction using both methods in which the IAC was incorporated. The methods enable a reduction in assay time to two days to test food samples, compared to five days required for the standardized procedures.To improve molecular methods to detect the pathogen in environmental substrates, five commercially available DNA extraction methods were evaluated in Chapter three with respect to DNA extraction efficiency of S. Enteriditis from soil, manure and compost. An internal procedural control (GIPC) for DNA extraction and amplification was developed. The GIPC was based on the same control DNA as used for the IAC in Chapter 1, incorporating gfp containing bacterial cells ( E. coli harboring a gfp-plasmid) in the sample prior to DNA extraction. Inclusion of the GIPC permitted a more accurate quantification of S. Enteriditis after DNA extraction and amplification and reduced the possibility of false-negatives. Using this protocol, the optimal extraction method differed forsoil (Mobio soil DNA extraction kit), manure (Bio101 soil DNA extraction kit) and compost (Mobio fecal DNA extraction kit).With each method, at least 2000 CFU of added S. Enteriditis/100 mg substrate could be detected bydirect DNA extraction and subsequent S. enterica specific Taqman PCR. After bacterial enrichment, as little as 1 CFU/100 mg of original substrate was detected. Using this approach a more reliable quantification was obtained for S. enterica initially present in environmental substrates.In Chapter four the physiological and molecular interactions between the human pathogenicS. Dublinand the commercially available mini Roman lettucecvTamburo are described. Investigation of the localization of S.Dublinon/in lettuce plants revealed the presence of significant populations on the surface and inside the plants. The latter was evidenced from significant residual concentrations after highly efficient surface disinfection (99.81%) and fluorescence microscopy ofS.Dublinin cross-sections of lettuce at the root-shoot transition region. A reduction in biomass was observed upon colonization of lettuce plants withS.Dublincompared to water-inoculated plants. Next to this physiological response, there were clear differential gene expression profiles between non-colonized and colonized lettuce plants based on transcriptome analysis by cDNA-AFLP. To confirm the results, generally and differentially expressed genes were selected, identified by sequence analysis and analyzed by RT-PCR to present the specific gene expression profiles in time. Functional grouping of the expressed genes indicated a correlation between colonization of the plants and an increase in expressed pathogenicity-related genes. From these results it was evident that lettuce plants respond to the presence ofS.Dublinat a physiological and molecular level. In addition, it was confirmed thatSalmonella serovars can colonize the interior of lettuce plants, thus potentially imposing a human health risk when contaminated lettuce is processed and consumed.The fact that the lettuce plants responded to the colonization by Salmonella serovars suggested that differences in susceptibility between cultivars or differences in colonization efficiency between Salmonella serovars might be present. In Chapter five, the differential interaction of S. Typhimurium, S. Enteritidis, S . Dublin, S. Newport and S. Montevideo with lettuce cultivars Cancan, Nelly and Tamburo is presented, in terms of prevalence and degree of endophytic colonization of lettuce by the Salmonella serovars. Besides a significant interaction, significant differences among serovars, but not among lettuce cultivars, were obtained when lettuce was grown under axenic conditions. When grown on soil, all three evaluated serovars S. Typhimurium, S. Enteritidis andS . Dublinwere able to colonize lettuce epiphytically, but to a lower extent than on axenically grown plants. OnlyS. Dublinwas able to colonize the plants endophytically when these were grown on contaminated soil. Species richness and diversity of the endophytic microbial community, determined from DGGE gels with DNA from Salmonella -colonized lettuce Cancan and Nelly, were negatively correlated with the number of Salmonella CFU / gram of lettuce. No correlation was observed for cultivar Tamburo. Thus, the microflora of lettuce cultivars Cancan and Nelly appeared more antagonistic to Salmonella serovars than that of cultivar Tamburo.Besides plant-associated colonization, also the active movement of Salmonella serovars towards lettuce roots was assessed. Movement was visualized using a metabolism marker (tetrazolium) for chemotaxis. Reduction of this marker suggested the presence of an organic compound in the lettuce root exudates that was used as carbon source by the Salmonella serovars. Subsequent micro-array analyses with DNA extracted from a broth culture of Salmonella with or without exudates identified genes of S. Typhimurium that were induced by root exudates. These genes, trehalose-6-phosphate synthase ( Ots A; utilizes glucose-6-phosphate as substrate), hexose phosphate utilization protein ( Uhp C; sensor for external glucose-6-phosphate), putative effector protein ( Ssa H; regulator of secretion of the type III secretion system),and putative anti-silencer RNA( Drs A; regulator of transcription to express rcs A promoter, responsible for capsular polysaccharide synthesis), imply a relation with a sugar-like carbon source and thus suggest an association with chemotaxis. The results described in Chapter 5 reveal different plant and microbial factors that influence the colonization efficiency of Salmonella serovars. The serovar and cultivar, but indirectly also the rhizosphere and the endophytic microflora of lettuce were most influential with respect to the risk of colonization and thus the risk for human health.Finally, an extensive discussion concerning the research of Chapters two to five is described in Chapter six, including future perspectives of risk for human health, route of infection and risk reduction in the production chain of Salmonella -associated lettuce

Comparison of real-time PCR methods for detection of Salmonella enterica and Escherichia coli O157:H7, and introduction of a general internal amplification control

The objectives of this study were to compare different real-time PCR-based methods for detection of either Salmonella spp. or E. coli O157:H7 with respect to sensitivity, precision and accuracy. In addition, a general internal amplification control (IAC) is presented, allowing prevention of false negative results. The IAC allows insight in amplification efficiency and enables a more accurate quantification with the evaluated real-time PCR methods. Implementation of the IAC with the different PCR methods did not affect the precision of the methods, but the sensitivity was reduced 10-fold. Introduction of an IAC with the Salmonella enterica specific detection method showed a shift in Ct-value (increase of target Ct-value with 0.45 +/- 0.17 cycles), while with the method to detect E. coli O157:H7 no influence of IAC co-amplification was observed. The quantification threshold of the methods in which the IAC was included was determined at 1 pg of target DNA (equal to 200 CFU) per reaction. Qualitative detection was feasible down to 10 fg of target DNA per reaction using both methods in which the IAC was incorporated. The adjusted methods have the potential to provide fast and sensitive detection of Salmonella spp. or E coli O157:H7, enabling accurate quantification and preventing false negative results by using the general IAC. (C) 2004 Elsevier B.V All rights reserved

Pathogenen in de primaire productieketen van bladgroenten

Wereldwijd is voedselvergiftiging een volkgezondheidsprobleem, meestal toegeschreven aan consumptie van besmette eieren of vlees. Echter, een deel wordt veroorzaakt door besmette rauwe groenten. Promovendus E. Franz onderzocht welke factoren van invloed zijn op de overleving van bacteriën tijdens de productie van bladgroente

Development of a multiplex AmpliDet RNA assay for simultaneous detection and typing of potato virus Y isolates

A multiplex AmpliDet RNA assay was developed for the specific detection of potato virus Y (PVY), and for the differentiation of the PVY(N), PVY(O/C) strains and the tuber necrotic isolates (PVY(NTN)). The assay is based on the generic amplification of a region within the coat protein coding region of all known PVY isolates by nucleic acid sequence-based amplification (NASBA) and strain-specific detection by molecular beacons. PVY(NTN)-specific diagnosis is achieved by detecting PVY(N) and PVY(O)-specific sequences flanking a recombination site that is associated with the tuber necrotic pathotype. The assay exhibited good specificity toward the various PVY strains in both single and mixed infections. The technique was validated by the use of 47 PVY isolates originating from six countries. The results of the AmpliDet RNA assay were identical or consistent with those of biological characterisation in the decisive majority of case

Development of a multiplex AmpliDet RNA assay for simultaneous detection and typing of potato virus Y isolates

A multiplex AmpliDet RNA assay was developed for the specific detection of potato virus Y (PVY), and for the differentiation of the PVY(N), PVY(O/C) strains and the tuber necrotic isolates (PVY(NTN)). The assay is based on the generic amplification of a region within the coat protein coding region of all known PVY isolates by nucleic acid sequence-based amplification (NASBA) and strain-specific detection by molecular beacons. PVY(NTN)-specific diagnosis is achieved by detecting PVY(N) and PVY(O)-specific sequences flanking a recombination site that is associated with the tuber necrotic pathotype. The assay exhibited good specificity toward the various PVY strains in both single and mixed infections. The technique was validated by the use of 47 PVY isolates originating from six countries. The results of the AmpliDet RNA assay were identical or consistent with those of biological characterisation in the decisive majority of case

Comparison of Methods of Extracting Salmonella enterica Serovar Enteritidis DNA from Environmental Substrates and Quantification of Organisms by Using a General Internal Procedural Control

This paper compares five commercially available DNA extraction methods with respect to DNA extraction efficiency of Salmonella enterica serovar Enteritidis from soil, manure, and compost and uses an Escherichia coli strain harboring a plasmid expressing green fluorescent protein as a general internal procedural control. Inclusion of this general internal procedural control permitted more accurate quantification of extraction and amplification of S. enterica serovar Enteritidis in these samples and reduced the possibility of false negatives. With this protocol it was found that the optimal extraction method differed for soil (Mobio soil DNA extraction kit), manure (Bio101 soil DNA extraction kit), and compost (Mobio fecal DNA extraction kit). With each method, as little as 1.2 × 10(3) to 1.8 × 10(3) CFU of added serovar Enteritidis per 100 mg of substrate could be detected by direct DNA extraction and subsequent S. enterica-specific TaqMan PCR. After bacterial enrichment, as little as 1 CFU/100 mg of original substrate was detected. Finally, the study presents a more accurate molecular analysis for quantification of serovar Enteritidis initially present in soil or manure using DNA extraction and TaqMan PCR

Prevalence of shiga toxin-producing Escherichia coli stx1, stx2, eaeA, and rfbE genes and survival of E. coli O157:H7 in manure from organic and low-input conventional dairy farms

Manure samples were collected from 16 organic (ORG) and 9 low-input conventional (LIC) Dutch dairy farms during August and September 2004 to determine the prevalence of the STEC virulence genes stx(1) (encoding Shiga toxin 1), stx(2) (encoding Shiga toxin 2), and eaeA (encoding intimin), as well as the rfbE gene, which is specific for Escherichia coli O157. The rfbE gene was present at 52% of the farms. The prevalence of rfbE was higher at ORG farms (61%) than at LIC farms (36%), but this was not significant. Relatively more LIC farms were positive for all Shiga toxin-producing E. coli (STEC) virulence genes eaeA, stx(1), and stx(2), which form a potentially highly virulent combination. Species richness of Enterobacteriaceae, as determined by DGGE, was significantly lower in manure positive for rfbE. Survival of a green fluorescent protein-expressing E. coli O157:H7 strain was studied in the manure from all farms from which samples were obtained and was modeled by a biphasic decline model. The time needed to reach the detection limit was predominantly determined by the level of native coliforms and the pH (both negative relationships). Initial decline was faster for ORG manure but leveled off earlier, resulting in longer survival than in LIC manure. Although the nonlinear decline curve could theoretically be explained as the cumulative distribution of an underlying distribution of decline kinetics, it is proposed that the observed nonlinear biphasic pattern of the survival curve is the result of changing nutrient status of the manure over time (and thereby changing competition pressure), instead of the presence of subpopulations differing in the level of resistance