Identification of Salmonella for public health surveillance using whole genome sequencing.

In April 2015, Public Health England implemented whole genome sequencing (WGS) as a routine typing tool for public health surveillance of Salmonella, adopting a multilocus sequence typing (MLST) approach as a replacement for traditional serotyping. The WGS derived sequence type (ST) was compared to the phenotypic serotype for 6,887 isolates of S. enterica subspecies I, and of these, 6,616 (96%) were concordant. Of the 4% (n = 271) of isolates of subspecies I exhibiting a mismatch, 119 were due to a process error in the laboratory, 26 were likely caused by the serotype designation in the MLST database being incorrect and 126 occurred when two different serovars belonged to the same ST. The population structure of S. enterica subspecies II-IV differs markedly from that of subspecies I and, based on current data, defining the serovar from the clonal complex may be less appropriate for the classification of this group. Novel sequence types that were not present in the MLST database were identified in 8.6% of the total number of samples tested (including S. enterica subspecies I-IV and S. bongori) and these 654 isolates belonged to 326 novel STs. For S. enterica subspecies I, WGS MLST derived serotyping is a high throughput, accurate, robust, reliable typing method, well suited to routine public health surveillance. The combined output of ST and serovar supports the maintenance of traditional serovar nomenclature while providing additional insight on the true phylogenetic relationship between isolates.

Salmonella enterica serovar Enteritidis, England and Wales, 1945-2011.

In England and Wales, the emergence of Salmonella enterica serovar Enteritidis resulted in the largest and most persistent epidemic of foodborne infection attributable to a single subtype of any pathogen since systematic national microbiological surveillance was established. We reviewed 67 years of surveillance data to examine the features, underlying causes, and overall effects of S. enterica ser. Enteritidis. The epidemic was associated with the consumption of contaminated chicken meat and eggs, and a decline in the number of infections began after the adoption of vaccination and other measures in production and distribution of chicken meat and eggs. We estimate that >525,000 persons became ill during the course of the epidemic, which caused a total of 6,750,000 days of illness, 27,000 hospitalizations, and 2,000 deaths. Measures undertaken to control the epidemic have resulted in a major reduction in foodborne disease in England and Wales.

A novel real-time polymerase chain reaction for identification of Salmonella enterica subspecies enterica.

Salmonella enterica subspecies enterica (subspecies I) causes the majority of infections in humans and homeothermic animals. We present a real-time polymerase chain reaction assay targeting the hilA gene that demonstrates 97.9% specificity and 99.9% sensitivity for rapid and reliable identification of subspecies I, offering savings in time and labor over traditional methods.

Rapid identification of Salmonella enterica subsp. arizonae and S. enterica subsp. diarizonae by real-time polymerase chain reaction.

Reptiles are popular as pets, leading to an increased risk of human infections due to uncommon Salmonella strains including the Arizona group (subspecies arizonae and diarizonae). We present a real-time Arizona-specific polymerase chain reaction demonstrating 100% specificity and 99.6% sensitivity, offering savings in time and labor over traditional identification methods.

Pulsed-field gel electrophoresis for molecular epidemiology of food pathogens.

Foodborne diseases due to well-recognized pathogens have emerged as an important and growing public health problem with a significant impact on health. Molecular methods for subtyping these microorganisms have become a valid adjunct to the traditional techniques employed in most laboratories. One such molecular technique for the detection and identification of food pathogens is pulsed-field gel electrophoresis (PFGE). This method separates large DNA molecules by the use of an alternating electrical field, such that greater size resolution can be obtained when compared to normal agarose gel electrophoresis. PFGE is often employed to track pathogens, such as Salmonella, Shigella, Escherichia coli (including O157), Campylobacter, and Listeria species through the food chain. The contour-clamped homogeneous electric field (CHEF) PFGE system is considered to be the gold standard for use in epidemiological studies of these organisms.

Flagellin gene sequence evolution in Salmonella.

Salmonella exhibits 70 serologically distinct flagellins, used internationally to diagnose and track infections. The terminal sequences of flagellin protein subunits are conserved in a range of bacteria and are here used as evolutionary markers to reveal how new serotypes arise. Terminal sequences of flagellins that exhibit factors g or m (G-group) were distinct from other Salmonella antigens (Non-G-group) and cluster more closely with Escherichia coli. It is postulated that G-group flagellins were inherited from a common ancestor of E. coli and Salmonella and that these antigens were among the original set in Salmonella. Sequence differences at the 5' termini may prevent recombination between co-infecting strains. Evidence of increased variation of flagellin in rare biphasic G-group serotypes suggests that the presence of a second flagellin locus allows mutation of the G-group flagellin. FljB probably arose from a single duplication of a Non-G gene, since which synonymous mutations resulted in the fljB-specific sequence at the 5' termini.

Towards the development of a DNA-sequence based approach to serotyping of Salmonella enterica.

BACKGROUND: The fliC and fljB genes in Salmonella code for the phase 1 (H1) and phase 2 (H2) flagellin respectively, the rfb cluster encodes the majority of enzymes for polysaccharide (O) antigen biosynthesis, together they determine the antigenic profile by which Salmonella are identified. Sequencing and characterisation of fliC was performed in the development of a molecular serotyping technique. RESULTS: FliC sequencing of 106 strains revealed two groups; the g-complex included those exhibiting "g" or "m,t" antigenic factors, and the non-g strains which formed a second more diverse group. Variation in fliC was characterised and sero-specific motifs identified. Furthermore, it was possible to identify differences in certain H antigens that are not detected by traditional serotyping. A rapid short sequencing assay was developed to target serotype-specific sequence motifs in fliC. The assay was evaluated for identification of H1 antigens with a panel of 55 strains. CONCLUSION: FliC sequences were obtained for more than 100 strains comprising 29 different H1 alleles. Unique pyrosequencing profiles corresponding to the H1 component of the serotype were generated reproducibly for the 23 alleles represented in the evaluation panel. Short read sequence assays can now be used to identify fliC alleles in approximately 97% of the 50 medically most important Salmonella in England and Wales. Capability for high throughput testing and automation give these assays considerable advantages over traditional methods.