In the United States, Salmonella
is the leading cause of bacterial food-borne disease, with approximately 1.4 million human cases each year since 1996 (42
). The second most-reported serovar of Salmonella
causing human disease is Salmonella enterica
serovar Enteritidis, which causes nearly as many cases of salmonellosis as Salmonella enterica
serovar Typhimurium, the most prevalent Salmonella
). The major food vehicle for S.
Enteritidis is shell eggs, as 80% of the S.
Enteritidis outbreaks and approximately 50,000 to 110,000 cases are egg associated in the United States each year (6
). In 2010 a large S.
Enteritidis outbreak was associated with consumption of shell eggs, with more than 1,800 reported cases (9
). Another common food vehicle for S.
Enteritidis is chicken meat (22
); however, other food vehicles have also been reported (30
Shell egg and chicken meat food systems are complex and contain a large number of niches that may be potential sources of S.
Enteritidis has been isolated from a wide variety of animals, such as rodents, wild birds, and insects, which could serve as potential sources (17
). Additional potential sources for S.
Enteritidis include chicken manure, sewage, and other moist and organic materials in farm environments (6
). Transmission of S.
Enteritidis to poultry can also occur via contaminated water and feed (6
). In densely populated poultry houses, the transmission of S.
Enteritidis among chickens occurs through direct contact with colonized birds or indirectly through contaminated materials (6
). This type of external contamination by feces and environments containing S.
Enteritidis is referred to as horizontal transmission (14
). Eggs can also become contaminated internally via vertical transmission when the ovaries of layers are colonized by S.
). In the past, most outbreaks of S.
Enteritidis traced back to flocks in New England, especially to states in the mid-Atlantic region, which includes the state of Pennsylvania (31
). However, while the total number of outbreaks in the northeastern United States decreased after 1996, the number in the western United States has increased since then (35
). The reduction in the number of egg-associated outbreaks in the northeastern United States has been attributed to egg quality assurance programs, such as the Pennsylvania Egg Quality Assurance Program (PEQAP), that attempt to reduce or eliminate sources of S
. Enteritidis contamination (31
). These programs typically involve acquisition of S.
Enteritidis-free chicks, control of pests (including rodents and insects), use of S.
Enteritidis-free feeds, flock vaccination, and routine microbiologic testing for S.
Enteritidis in the production environment (6
Several molecular subtyping methods have been developed for studying the epidemiology of S.
Enteritidis, including amplified fragment length polymorphism (AFLP) (15
), multiple-locus variable-number tandem-repeat analysis (MLVA) (3
), and pulsed-field gel electrophoresis (PFGE) (18
). PFGE is currently the “gold standard” method used by public health surveillance laboratories for tracking food-borne pathogens, including Salmonella
). The main advantage of PFGE is its normally high discriminatory power (i.e., ability to separate unrelated strains) for subtyping most serovars of Salmonella
. However, PFGE lacks discriminatory power for highly clonal serovars like S.
). For example, the most recent S.
Enteritidis outbreak due to shell eggs was caused by the most common PFGE pattern for S.
Enteritidis in the PulseNet database (JEGX01.0004); thus, not all isolates with this pulsotype may be related to this outbreak (9
). This lack of adequate discriminatory power makes it difficult to track a specific clone of S.
Enteritidis in the food system using PFGE. Besides occasional inadequate discriminatory power, PFGE does not provide appropriate information to infer phylogenetic relationships among subtypes (34
). Another subtyping method, MLVA, was reported to have higher discriminatory power than PFGE for S.
). However, in some circumstances, strains that had the same MLVA type were separated by PFGE (5
). Moreover, replicates of the same strains of Salmonella
have been shown to have different numbers of repeat units at a specific locus (7
), which makes accurate interpretation of results difficult.
Compared to PFGE and MLVA, MLST, which targets nucleotide sequence differences in several DNA loci, generates discrete, highly informative, highly portable, and reproducible data. Moreover, MLST is a well-accepted tool for studying the population structure, evolution, and diversity of bacteria (25
). Recently, a new sequence typing scheme based on virulence genes (fimH
) and clustered regularly interspaced short palindromic repeats (CRISPRs) (here designated CRISPR-MVLST) was shown to provide better identification and separation of S.
Enteritidis outbreak strains/clones than PFGE (29
). Virulence genes were congruent with serotyping and appeared to differentiate epidemic clones within different serotypes of Salmonella
, while CRISPRs appeared to differentiate outbreak strains/clones within epidemic clones. CRISPRs encode tandem sequences containing 21- to 47-bp direct repeats (DRs) and spacers of similar size (see Fig. S1 in the supplemental material). Spacers are short DNA sequences obtained from foreign nucleic acids, such as phages or plasmids, that are inserted into bacterial chromosomes to protect them from infection by homologous phages and plasmids (2
). Therefore, different CRISPRs arise due to diverse phage and plasmid pools in the environment and therefore contain ecologic and geographic information specific to the bacteria present there (24
). This might explain the uneven distribution of Salmonella
subtypes from different sources, as observed previously (36
). Source attribution is especially critical in the case of S.
Enteritidis, because as mentioned above, this is currently difficult using current subtyping methods.
Therefore, the purpose of the present study was to utilize CRISPR-MVLST to subtype S. Enteritidis isolates from different sources, including clinical, poultry, egg, and environmental sources.