Identification of Genes Associated with Survival of Salmonella enterica Serovar Enteritidis in Chicken Egg Albumen

Salmonella enterica consists of over 2,000 serovars that are major causes of morbidity and mortality associated with contaminated food. Despite similarities among serovars of Salmonella enterica, many demonstrate unique host specificities, epidemiological characteristics, and clinical manifestations. One of the unique epidemiological characteristics of the serovar Enteritidis is that it is the only bacterium routinely transmitted to humans through intact chicken eggs. Therefore, Salmonella enterica serovar Enteritidis must be able to persist inside chicken eggs to be transmitted to humans, and its survival in egg is important for its transmission to the human population. The ability of Salmonella enterica serovar Enteritidis to survive in and transmit through eggs may have contributed to its drastically increased prevalence in the 1980s and 1990s. In the present study, using transposon-mediated mutagenesis, we have identified genes important for the association of Salmonella enterica serovar Enteritidis with chicken eggs. Our results indicate that genes involved in cell wall structural and functional integrity, and nucleic acid and amino acid metabolism are important for Salmonella enterica serovar Enteritidis to persist in egg albumen. Two regions unique to Salmonella enterica serovar Enteritidis were also identified, one of which enhanced the survival of a Salmonella enterica serovar Typhimurium isolate in egg albumen. The implication of our results to the serovar specificity of Salmonella enterica is also explored in the present study.     

Bacteriophage Therapy To Reduce Salmonella Colonization of Broiler Chickens

Acute enteric infections caused by salmonellas remain a major public health burden worldwide. Poultry, particularly chickens, are known to be the main reservoir for this zoonotic pathogen. Although some progress has been made in reducing Salmonella colonization of broiler chickens by using biosecurity and antimicrobials, it still remains a considerable problem. The use of host-specific bacteriophages as a biocontrol is one possible intervention by which Salmonella colonization could be reduced. A total of 232 Salmonella bacteriophages were isolated from poultry farms, abattoirs, and wastewater in 2004 and 2005. Three phages exhibiting the broadest host ranges against Salmonella enterica serotypes Enteritidis, Hadar, and Typhimurium were characterized further by determining their morphology and lytic activity in vitro. These phages were then administered in antacid suspension to birds experimentally colonized with specific Salmonella host strains. The first phage reduced S. enterica serotype Enteritidis cecal colonization by ≥4.2 log₁₀ CFU within 24 h compared with controls. Administration of the second phage reduced S. enterica serotype Typhimurium by ≥2.19 log₁₀ CFU within 24 h. The third bacteriophage was ineffective at reducing S. enterica serotype Hadar colonization. Bacteriophage resistance occurred at a frequency commensurate with the titer of phage being administered, with larger phage titers resulting in a greater proportion of resistant salmonellas. The selection of appropriate bacteriophages and optimization of both the timing and method of phage delivery are key factors in the successful phage-mediated control of salmonellas in broiler chickens.     

Specific Discrimination of Three Pathogenic Salmonella enterica subsp. enterica Serotypes by carB-Based Oligonucleotide Microarray

It is important to rapidly and selectively detect and analyze pathogenic Salmonella enterica subsp. enterica in contaminated food to reduce the morbidity and mortality of Salmonella infection and to guarantee food safety. In the present work, we developed an oligonucleotide microarray containing duplicate specific capture probes based on the carB gene, which encodes the carbamoyl phosphate synthetase large subunit, as a competent biomarker evaluated by genetic analysis to selectively and efficiently detect and discriminate three S. enterica subsp. enterica serotypes: Choleraesuis, Enteritidis, and Typhimurium. Using the developed microarray system, three serotype targets were successfully analyzed in a range as low as 1.6 to 3.1 nM and were specifically discriminated from each other without nonspecific signals. In addition, the constructed microarray did not have cross-reactivity with other common pathogenic bacteria and even enabled the clear discrimination of the target Salmonella serotype from a bacterial mixture. Therefore, these results demonstrated that our novel carB-based oligonucleotide microarray can be used as an effective and specific detection system for S. enterica subsp. enterica serotypes.     

Plasmid Profile and Pulsed–Field Gel Electrophoresis Analysis of Salmonella enterica Isolates from Humans in Turkey

This study was conducted for typing Salmonella enterica subspecies enterica strains in Turkey using pulsed–field gel electrophoresis (PFGE) and plasmid DNA profile analysis. Fourty-two strains were isolated from clinical samples obtained from unrelated patients with acute diarrhea. The samples were collected from state hospitals and public health laboratories located at seven provinces in different regions of Turkey at different times between 2004 and 2010. The strains were determined to belong to 4 different serovars. The Salmonella enterica strains belonged to the serovars Salmonella Enteritidis (n = 23), Salmonella Infantis (n = 14), Salmonella Munchen (n = 2), and Salmonella Typhi (n = 3). Forty-two Salmonella enterica strains were typed with PFGE methods using XbaI restriction enzyme and plasmid analysis. At the end of typing, 11 different PFGE band profiles were obtained. Four different PFGE profiles (type 1, 4, 9, and 10) were found among serotype S. Enteritidis species, 3 different PFGE profiles (type 3, 5, 6) were found among S. Infantis species, 2 different PFGE profiles were found among S. Typhi species (type 2 and 11), and 2 different PFGE profiles were found among S. Munchen species (type 7, 8). The UPGMA dendrogram was built on the PFGE profiles. In this study, it was determined that 4 strains of 42 Salmonella enterica strains possess no plasmid, while the isolates have 1–3 plasmids ranging from 5.0 to 150 kb and making 12 different plasmid profiles (P1–P12). In this study, we have applied the analysis of the PFGE patterns and used bioinformatics methods to identify both inter and intra serotype relationships of 4 frequently encountered serotypes for the first time in Turkey.     

Microarray-Based Detection of Salmonella enterica Serovar Enteritidis Genes Involved in Chicken Reproductive Tract Colonization

Salmonella enterica serovar Enteritidis has developed the potential to contaminate table eggs internally, by colonization of the chicken reproductive tract and internalization in the forming egg. The serotype Enteritidis has developed mechanisms to colonize the chicken oviduct more successfully than other serotypes. Until now, the strategies exploited by Salmonella Enteritidis to do so have remained largely unknown. For that reason, a microarray-based transposon library screen was used to identify genes that are essential for the persistence of Salmonella Enteritidis inside primary chicken oviduct gland cells in vitro and inside the reproductive tract in vivo. A total of 81 genes with a potential role in persistence in both the oviduct cells and the oviduct tissue were identified. Major groups of importance include the Salmonella pathogenicity islands 1 and 2, genes involved in stress responses, cell wall, and lipopolysaccharide structure, and the region-of-difference genomic islands 9, 21, and 40.     

Development of a Cell Culture Method To Isolate and Enrich Salmonella enterica Serotype Enteritidis from Shell Eggs for Subsequent Detection by Real-Time PCR

Salmonella enterica serotype Enteritidis is a major cause of nontyphoidal salmonellosis from ingestion of contaminated raw or undercooked shell eggs. Current techniques used to identify Salmonella serotype Enteritidis in eggs are extremely laborious and time-consuming. In this study, a novel eukaryotic cell culture system was combined with real-time PCR analysis to rapidly identify Salmonella serotype Enteritidis in raw shell eggs. The system was compared to the standard microbiological method of the International Organization for Standardization (Anonymous, Microbiology of food and animal feeding stuffs—horizontal method for the detection of Salmonella, 2002). The novel technique utilizes a mouse macrophage cell line (RAW 264.7) as the host for the isolation and intracellular replication of Salmonella serotype Enteritidis. Exposure of macrophages to Salmonella serotype Enteritidis-contaminated eggs results in uptake and intracellular replication of the bacterium, which can subsequently be detected by real-time PCR analysis of the DNA released after disruption of infected macrophages. Macrophage monolayers were exposed to eggs contaminated with various quantities of Salmonella serotype Enteritidis. As few as 10 CFU/ml was detected in cell lysates from infected macrophages after 10 h by real-time PCR using primer and probe sets specific for DNA segments located on the Salmonella serotype Enteritidis genes sefA and orgC. Salmonella serotype Enteritidis could also be distinguished from other non-serogroup D Salmonella serotypes by using the sefA- and orgC-specific primer and probe sets. Confirmatory identification of Salmonella serotype Enteritidis in eggs was also achieved by isolation of intracellular bacteria from lysates of infected macrophages on xylose lysine deoxycholate medium. This method identifies Salmonella serotype Enteritidis from eggs in less than 10 h compared to the more than 5 days required for the standard reference microbiological method of the International Organization for Standardization (Microbiology of food and animal feeding stuffs—horizontal method for the detection of Salmonella, 2002).Nontyphoidal salmonellosis is an invasive intestinal disease contracted predominately by ingestion of food contaminated with serotypes of the gram-negative bacterial species Salmonella enterica. Gastroenteritis caused by Salmonella spp. represents a large portion of the natural food-borne illnesses that occur worldwide each year. Bacterial virulence is established in part by the bacterium''s ability to invade and survive within host cells (20). S. enterica is capable of survival within a wide array of host cells, including epithelial cells, dendritic cells, and macrophages in both animal and cell culture models (16, 17, 18, 19). However, survival in macrophages is required for initiation of systemic infection (24). Two chromosomal pathogenicity islands, SPI-1 and SPI-2, which are present in all Salmonella enterica serotypes, are essential for the invasion of epithelial cells and intracellular replication in macrophages, respectively (13, 14).There are currently over 2,500 distinct serotypes of S. enterica (http://www.pasteur.fr/sante/clre/cadrecnr/salmoms/WKLM_2007.pdf). Of these, Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium are most commonly associated with food-borne illness in humans (4). Raw and undercooked shell eggs have been implicated as vehicles for the transmission of both of these serotypes of Salmonella enterica (9, 38). However, Salmonella serotype Enteritidis infection has been more frequently linked to shell egg consumption, whereas Salmonella serotype Typhimurium infection is more often associated with the consumption of contaminated chicken meat (8). Of the 309 documented outbreaks of Salmonella serotype Enteritidis in the United States from 1990 to 2001, 241 were attributed to the consumption of raw or undercooked eggs (6). Salmonella serotype Enteritidis phage types 4, 8, and 13 have been implicated in the majority of salmonellosis cases from the consumption of egg products (5). In addition, Salmonella serotype Enteritidis is able to colonize laying hen reproductive organs and developing eggs and has been shown to persist in eggs after they have been laid (23).A variety of methods have been developed in order to expedite the detection of salmonellae in eggs, including GeneQuence DNA hybridization, PCR analysis, and enzyme-linked immunosorbent assay (3, 27, 37). However, these methods require lengthy enrichment steps prior to the application of the respective methods. Real-time PCR (RT-PCR) is a promising new method currently used for detection of a wide variety of bacterial pathogens in food matrices (12, 15, 22, 34, 40). However, this technique can be ineffective for the detection of Salmonella serotype Enteritidis in foods such as eggs due to the presence of PCR-inhibitory components (41).In this study, we developed a novel detection system to allow for the specific identification of viable Salmonella serotype Enteritidis in raw shell eggs. The method developed is based on the ability of Salmonella to invade and replicate within macrophages as part of its life cycle within a host. In theory, cultured eukaryotic cell lines exposed to Salmonella-contaminated foods will allow the penetration and replication of Salmonella while confining food particles and noninvasive bacteria to the extracellular environment, allowing the isolation and enrichment of intracellular Salmonella for subsequent detection by commercially available techniques, such as RT-PCR. In practice, a suitable mammalian cell monolayer is exposed to a particular food matrix suspected of harboring salmonellae. The exposure is promoted for sufficient time to allow cell contact and engulfment of salmonellae. The mammalian cell monolayer is then washed sufficiently to remove the food matrix and extracellular microorganisms. The infected cell monolayer is reconstituted with fresh medium and further incubated to allow for intracellular multiplication of Salmonella (postinfection). After the infection is terminated, the culture medium is discarded, the infected cells are disrupted, and the DNA present in the resultant lysates is analyzed by RT-PCR using primers and probes specific for unique Salmonella DNA sequences. We utilized this method for the presumptive and confirmatory identification of Salmonella serotype Enteritidis in raw shell eggs.     

Allele distribution and genetic diversity of VNTR loci in <Emphasis Type="Italic">Salmonella enterica</Emphasis>serotype Enteritidis isolates from different sources

Background  

Salmonella enterica serotype Enteritidis (S. Enteritidis) is a zoonotic pathogen, which can be found in many sources including animals and the environment. However, little is known about the molecular relatedness among S. Enteritidis isolates from different sources. We have applied multiple-locus variable number tandem repeat analysis (MLVA) to study the genetic diversity of S. Enteritidis isolates from human and non-human sources.     

Inhibition of Salmonella enterica Cells in Deli-Type Salad by Enterocin AS-48 in Combination with Other Antimicrobials

The cyclic antibacterial peptide enterocin AS-48 acted synergistically with p-hydroxybenzoic acid methyl ester (PHBME) and with 2-nitropropanol against Salmonella enterica serovar Enteritidis CECT 4300 in Russian salad. In challenge tests on a cocktail of Salmonella strains (S. enterica ssp. enterica serotype Typhi CECT 409, S. enterica ssp. enterica serovar Choleraesuis CECT 915, S. enterica ssp. enterica serovar Enteritidis CECT 4300, S. enterica ssp. arizonae serovar Arizonae CECT 4395, and S. enterica ssp. salamae CECT 4000) in salad at 10°C, the combinations of PHBME and AS-48 (80 μg/g) or 2-nitropropanol and AS-48 (40 μg/g) reduced the concentrations of viable Salmonella from 4.27 to 4.75 log CFU/g down to the limit of detection for 7 days. Salmonella populations did not increase in control samples (without any antimicrobials or in the presence of AS-48 alone) probably due to the low pH of this type of salad and low temperature of incubation, but retained over 85% viability after 1 week. This work opens new possibilities to expand the spectrum of inhibition of enterocin AS-48 against Salmonella enterica.     

Public Health Assessment of Salmonella enterica Serovar Enteritidis Inactivated-Vaccine Treatment in Layer Flocks

Although there have been several reports on the efficacy assessment of a Salmonella enterica serovar Enteritidis vaccine against intestinal and parenchymatous organ diseases of laying hens, no public health risk characterization of its long-term effect on eggs has been reported. In this study, we attempted to assess the public health effect of an inactivated S. enterica serovar Enteritidis vaccine against serovar Enteritidis contamination of chicken eggs. We analyzed serovar Enteritidis isolation test results from four windowless farms in which inactivated-vaccine administration was initiated based on the sanitary monitoring program of a farm. When flocks with and without S. enterica serovar Enteritidis vaccine treatments were mixed, the application of an inactivated serovar Enteritidis vaccine decreased the most probable number (MPN) of bacteria by at least 100-fold in broken (liquid) egg samples positive for serovar Enteritidis, although a statistical difference between those MPNs could not be obtained. The isolation frequency after the vaccine application was less than 1/10 (P < 0.01). No S. enterica serovar Enteritidis bacteria were isolated approximately 1 year after all of the chickens had received the inactivated serovar Enteritidis vaccine. It was suggested that an adequate administration of an inactivated serovar Enteritidis vaccine reduced the contamination risk of eggs (the number of isolated serovar Enteritidis cells and detection frequency) compared to the contamination risk of eggs laid by nonvaccinated hens.     

Salmonella enterica Serovar Enteritidis Genes Induced during Oviduct Colonization and Egg Contamination in Laying Hens

Salmonella enterica serovar Enteritidis is the predominant serovar associated with salmonellosis worldwide, which is in part due to its ability to contaminate the internal contents of the hen's egg. It has been shown that S. enterica serovar Enteritidis has an unusual tropism for the avian reproductive tract and an ability to persist in the oviduct and ovary. Factors allowing S. enterica serovar Enteritidis strains to contaminate eggs could be a specific interaction with the oviduct tissue, leading to persisting oviduct colonization. In vivo expression technology, a promoter-trap strategy, was used to identify genes expressed during oviduct colonization and egg contamination with S. enterica serovar Enteritidis. A total of 25 clones with in vivo-induced promoters were isolated from the oviduct tissue and from laid eggs. Among the 25 clones, 7 were isolated from both the oviducts and the eggs. DNA sequencing of the cloned promoters revealed that genes involved in amino acid and nucleic acid metabolism, motility, cell wall integrity, and stress responses were highly expressed in the reproductive tract tissues of laying hens.     

Draft Genome Sequences of 21 Salmonella enterica Serovar Enteritidis Strains

Salmonella enterica subsp. enterica serovar Enteritidis is a common food-borne pathogen, often associated with shell eggs and poultry. Here, we report draft genomes of 21 S. Enteritidis strains associated with or related to the U.S.-wide 2010 shell egg recall. Eleven of these genomes were from environmental isolates associated with the egg outbreak, and 10 were reference isolates from previous years, unrelated to the outbreak. The whole-genome sequence data for these 21 human pathogen strains are being released in conjunction with the newly formed 100K Genome Project.     

Evaluation of DNA Extraction Methods for Use in Combination with SYBR Green I Real-Time PCR To Detect Salmonella enterica Serotype Enteritidis in Poultry

The objective of this study was to develop a rapid, reproducible, and robust method for detecting Salmonella enterica serotype Enteritidis in poultry samples. First, for the extraction and purification of DNA from the preenrichment culture, four methods (boiling, alkaline lysis, Nucleospin, and Dynabeads DNA Direct System I) were compared. The most effective method was then combined with a real-time PCR method based on the double-stranded DNA binding dye SYBR Green I used with the ABI Prism 7700 system. The specificity of the reaction was determined by the melting temperature (T_m) of the amplicon obtained. The experiments were conducted both on samples of chicken experimentally contaminated with serotype Enteritidis and on commercially available poultry samples, which were also used for comparisons with the standard cultural method (i.e., ISO 6579/2001). The results of comparisons among the four DNA extraction methods showed significant differences except for the results from the boiling and Nucleospin methods (the two methods that produced the lowest threshold cycles). Boiling was selected as the preferred extraction method because it is the simplest and most rapid. This method was then combined with SYBR Green I real-time PCR, using primers SEFA-1 and SEFA-2. The specificity of the reaction was confirmed by the T_m, which was consistently specific for the amplicon obtained; the mean peak T_m obtained with curves specific for serotype Enteritidis was 82.56 ± 0.22°C. The standard curve constructed using the mean threshold cycle and various concentrations of serotype Enteritidis (ranging from 10³ to 10⁸ CFU/ml) showed good linearity (R² = 0.9767) and a sensitivity limit of less than 10³ CFU/ml. The results of this study demonstrate that the SYBR Green I real-time PCR constitutes an effective and easy-to-perform method for detecting serotype Enteritidis in poultry samples.     

Application of Rapid Dot Blot Immunoassay for Detection of Salmonella enterica Serovar Enteritidis in Eggs, Poultry, and Other Foods

Salmonella enterica serovar Enteritidis was detected in artificially inoculated eggs within 24 h through a rapid monoclonal antibody-based dot blot immunoassay. Detection in poultry and other products required 28 h. Samples were directly enriched in homogenized egg without the need for pre- or postenrichment steps. Serovar Enteritidis was detected in the presence of other bacteria when outcompeted 1:400.     

Genome sequence analysis of 91 Salmonella Enteritidis isolates from mice caught on poultry farms in the mid 1990s

A total of 91 draft genome sequences were used to analyze isolates of Salmonella enterica serovar Enteritidis obtained from feral mice caught on poultry farms in Pennsylvania. One objective was to find mutations disrupting open reading frames (ORFs) and another was to determine if ORF-disruptive mutations were present in isolates obtained from other sources. A total of 83 mice were obtained between 1995–1998. Isolates separated into two genomic clades and 12 subgroups due to 742 mutations. Nineteen ORF-disruptive mutations were found, and in addition, bigA had exceptional heterogeneity requiring additional evaluation. The TRAMS algorithm detected only 6 ORF disruptions. The sefD mutation was the most frequently encountered mutation and it was prevalent in human, poultry, environmental and mouse isolates. These results confirm previous assessments of the mouse as a rich source of Salmonella enterica serovar Enteritidis that varies in genotype and phenotype.     

Exposure to a dog elicits different cardiovascular and behavioral effects in pregnant and lactating goats

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) was responsible for a worldwide pandemic during the 1980s and 1990s; however, changes in the dominant lineage before and after this event remain unknown. This study determined S. Enteritidis lineages before and after this pandemic event in Japan using multilocus sequence typing (MLST). Thirty S. Enteritidis strains were collected in Japan between 1973 and 2004, consisting of 27 human strains from individual episodes, a bovine strain, a liquid egg strain and an eggshell strain. Strains showed nine phage types and 17 pulsed-field profiles with pulsed-field gel electrophoresis. All strains had homologous type 11 sequences without any nucleotide differences in seven housekeeping genes. These MLST results suggest that S. Enteritidis with the diversities revealed by phage typing and pulsed-field profiling has a highly clonal population. Although type 11 S. Enteritidis may exhibit both pleiotropic surface structure and pulsed-field type variation, it is likely to be a stable lineage derived from an ancestor before the 1980s and/or 1990s pandemic in Japan.     

Long-Term Survival of Pathogenic and Sanitation Indicator Bacteria in Experimental Biowaste Composts

For economic, agricultural, and environmental reasons, composting is frequently used for organic waste recycling. One approach to limiting the potential risk from bacterial food-borne illnesses is to ensure that soil amendments and organic fertilizers are disinfected. However, more knowledge concerning the microbiological safety of composted substrates other than sludge and manure is necessary. Experimental in-vessel biowaste composts were used to study the survival of seeded Listeria monocytogenes, Salmonella enterica subsp. enterica serotype Enteritidis, and Escherichia coli. Four organic waste mixtures, containing various proportions of paper and cardboard, fruits and vegetables, and green waste, were composted in laboratory reactors with forced aeration. The physicochemical and microbiological parameters were monitored for 12 weeks during composting. The survival of bacteria over a 3-month period at 25°C was assessed with samples collected after different experimental composting times. Strain survival was also monitored in mature sterilized composts. Nonsterile composts did not support pathogen growth, but survival of seeded pathogens was observed. Salmonella serovar Enteritidis survived in all composts, and longer survival (3 months) was observed in mature composts (8 and 12 weeks of composting). Mature biowaste composts may support long-term survival of Salmonella serovar Enteritidis during storage at room temperature. E. coli and L. monocytogenes survival was observed only in 4-week-old composts and never in older composts. Proper composting may prevent long-term survival of E. coli and L. monocytogenes. These results suggest that like composted sewage sludge or manure, domestic waste composts may support pathogen survival. Survival was not related to the physicochemical characteristics of the composts.     

Uptake and Replication of Salmonella enterica in Acanthamoeba rhysodes

The ability of salmonellae to become internalized and to survive and replicate in amoebae was evaluated by using three separate serovars of Salmonella enterica and five different isolates of axenic Acanthamoeba spp. In gentamicin protection assays, Salmonella enterica serovar Dublin was internalized more efficiently than Salmonella enterica serovar Enteritidis or Salmonella enterica serovar Typhimurium in all of the amoeba isolates tested. The bacteria appeared to be most efficiently internalized by Acanthamoeba rhysodes. Variations in bacterial growth conditions affected internalization efficiency, but this effect was not altered by inactivation of hilA, a key regulator in the expression of the invasion-associated Salmonella pathogenicity island 1. Microscopy of infected A. rhysodes revealed that S. enterica resided within vacuoles. Prolonged incubation resulted in a loss of intracellular bacteria associated with morphological changes and loss of amoebae. In part, these alterations were associated with hilA and the Salmonella virulence plasmid. The data show that Acanthamoeba spp. can differentiate between different serovars of salmonellae and that internalization is associated with cytotoxic effects mediated by defined Salmonella virulence loci.     

Identification by Subtractive Hybridization of Sequences Specific for Salmonella enterica Serovar Enteritidis

Salmonella enterica serovar Enteritidis, a major cause of food poisoning, can be transmitted to humans through intact chicken eggs when the contents have not been thoroughly cooked. Infection in chickens is asymptomatic; therefore, simple, sensitive, and specific detection methods are crucial for efforts to limit human exposure. Suppression subtractive hybridization was used to isolate DNA restriction fragments present in Salmonella serovar Enteritidis but absent in other bacteria found in poultry environments. Oligonucleotide primers to candidate regions were used in polymerase chain reactions to test 73 non-Enteritidis S. enterica isolates comprising 34 different serovars, including Dublin and Pullorum, two very close relatives of Enteritidis. A primer pair to one Salmonella difference fragment (termed Sdf I) clearly distinguished serovar Enteritidis from all other serovars tested, while two other primer pairs only identified a few non-Enteritidis strains. These primer pairs were also useful for the detection of a diverse collection of clinical and environmental Salmonella serovar Enteritidis isolates. In addition, five bacterial genera commonly found with Salmonella serovar Enteritidis were not detected. By treating total DNA with an exonuclease that degrades sheared chromosomal DNA but not intact circular plasmid DNA, it was shown that Sdf I is located on the chromosome. The Sdf I primers were used to screen a Salmonella serovar Enteritidis genomic library and a unique 4,060-bp region was defined. These results provide a basis for developing a rapid, sensitive, and highly specific detection system for Salmonella serovar Enteritidis and provide sequence information that may be relevant to the unique characteristics of this serovar.     

Phenotypic and genotypic typing of<Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Enteritidis isolates from poultry farms environments in Tunisia

Salmonella enterica serovar Enteritidis is one of the majorSalmonella serovars which may cause animal infections and human salmonellosis. In this study, two hundred forty five samples (faeces, water and environmental swabs) were taken from eight poultry farms localized in different geographical areas of Tunisia. We foundSalmonella serovar Enteritidis (16 strains),Salmonella typhimirium (2),Salmonella scharzengrund (2), andSalmonella braenderup (1).Salmonella Enteritidis strains were characterized by pulsed field gel electrophoresis (PFGE) analysis, plasmid analysis and antibiotic resistance profiles.XbaI PFGE analysis revealed two PFGE types and plasmid profiling identified four plasmid types. The majority of isolates were susceptible to all antibiotic tested. The combined use of phenotypic and genotypic methods indicates the spread of a particularSalmonella Enteritidis clone. This clone is highly related to a major world-wide clone identified in many other countries.     

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 × 10³ to 1.8 × 10³ 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.