Nikki Shariat

CRISPRs: Molecular Signatures Used for Pathogen Subtyping

ABSTRACT Rapid and accurate strain identification is paramount in the battle against microbial outbreaks, and several subtyping approaches have been developed. One such method uses clustered regular interspaced short palindromic repeats (CRISPRs), DNA repeat elements that are present in approximately half of all bacteria. Though their signature function is as an adaptive immune system against invading DNA such as bacteriophages and plasmids, CRISPRs also provide an excellent framework for pathogen tracking and evolutionary studies. Analysis of the spacer DNA sequences that reside between the repeats has been tremendously useful for bacterial subtyping during molecular epidemiological investigations. Subtyping, or strain identification, using CRISPRs has been employed in diverse Gram-positive and Gram-negative bacteria, including Mycobacterium tuberculosis, Salmonella enterica, and the plant pathogen Erwinia amylovora. This review discusses the several ways in which CRISPR sequences are exploited for subtyping. This includes the well-established spoligotyping methodologies that have been used for 2 decades to type Mycobacterium species, as well as in-depth consideration of newer, higher-throughput CRISPR-based protocols.

The combination of CRISPR-MVLST and PFGE provides increased discriminatory power for differentiating human clinical isolates of Salmonella enterica subsp. enterica serovar Enteritidis

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) is a major cause of foodborne salmonellosis. Rapid, efficient and accurate methods for identification are required to track specific strains of S. Enteritidis during outbreaks of human salmonellosis. By exploiting the hypervariable nature of virulence genes and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs), we previously developed a powerful sequence-based subtyping approach, designated CRISPR-MVLST. To substantiate the applicability of CRISPR-MVLST, we analyzed a broad set of S. Enteritidis isolates collected over a six-year period. Among 141 isolates we defined 22 Enteritidis Sequence Types (ESTs), the majority of which were novel. Notably, strains exhibiting the common PFGE pattern, JEGX01.0004 (characteristic of ∼40% of S. Enteritidis isolates in the United States), were separated into twelve distinct sequence types. Conversely, isolates of EST4, the most predominant EST we observed, comprised eight different PFGE patterns. Importantly, we showed that some genotypes that were previously associated with the food supply chain at the farm level have now been identified in clinical samples. CRISPR sequence data shows subtle but distinct differences among different alleles of S. Enteritidis, suggesting that evolution of these loci occurs vertically, as opposed to previously reported evolution by spacer acquisition in other bacteria.

Comparative Analysis of Subtyping Methods against a Whole-Genome-Sequencing Standard for Salmonella enterica Serotype Enteritidis

ABSTRACT A retrospective investigation was performed to evaluate whole-genome sequencing as a benchmark for comparing molecular subtyping methods for Salmonella enterica serotype Enteritidis and survey the population structure of commonly encountered S. enterica serotype Enteritidis outbreak isolates in the United States. A total of 52 S. enterica serotype Enteritidis isolates representing 16 major outbreaks and three sporadic cases collected between 2001 and 2012 were sequenced and subjected to subtyping by four different methods: (i) whole-genome single-nucleotide-polymorphism typing (WGST), (ii) multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA), (iii) clustered regularly interspaced short palindromic repeats combined with multi-virulence-locus sequence typing (CRISPR-MVLST), and (iv) pulsed-field gel electrophoresis (PFGE). WGST resolved all outbreak clusters and provided useful robust phylogenetic inference results with high epidemiological correlation. While both MLVA and CRISPR-MVLST yielded higher discriminatory power than PFGE, MLVA outperformed the other methods in delineating outbreak clusters whereas CRISPR-MVLST showed the potential to trace major lineages and ecological origins of S. enterica serotype Enteritidis. Our results suggested that whole-genome sequencing makes a viable platform for the evaluation and benchmarking of molecular subtyping methods.

CRISPR-MVLST subtyping of Salmonella enterica subsp. enterica serovars Typhimurium and Heidelberg and application in identifying outbreak isolates

BackgroundSalmonella enterica subsp. enterica serovars Typhimurium (S. Typhimurium) and Heidelberg (S. Heidelberg) are major causes of foodborne salmonellosis, accounting for a fifth of all annual salmonellosis cases in the United States. Rapid, efficient and accurate methods for identification are required for routine surveillance and to track specific strains during outbreaks. We used Pulsed-field Gel Electrophoresis (PFGE) and a recently developed molecular subtyping approach termed CRISPR-MVLST that exploits the hypervariable nature of virulence genes and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) to subtype clinical S. Typhimurium and S. Heidelberg isolates.ResultsWe analyzed a broad set of 175 S. Heidelberg and S. Typhimurium isolates collected over a five-year period. We identified 21 Heidelberg Sequence Types (HSTs) and 37 Typhimurium STs (TSTs) that were represented by 27 and 45 PFGE pulsotypes, respectively, and determined the discriminatory power of each method.ConclusionsFor S. Heidelberg, our data shows that combined typing by both CRISPR-MVLST and PFGE provided a discriminatory power of 0.9213. Importantly, CRISPR-MVLST was able to separate common PFGE patterns such as JF6X01.0022 into distinct STs, thus providing significantly greater discriminatory power. Conversely, we show that subtyping by either CRISPR-MVLST or PFGE independently provides a sufficient discriminatory power (0.9345 and 0.9456, respectively) for S. Typhimurium. Additionally, using isolates from two S. Typhimurium outbreaks, we demonstrate that CRISPR-MVLST provides excellent epidemiologic concordance.

Subtyping of Salmonella enterica serovar Newport outbreak isolates by CRISPR-MVLST and determination of the relationship between CRISPR-MVLST and PFGE results.

ABSTRACT Salmonella enterica subsp. enterica serovar Newport (S. Newport) is the third most prevalent cause of food-borne salmonellosis. Rapid, efficient, and accurate methods for identification are required to track specific strains of S. Newport during outbreaks. By exploiting the hypervariable nature of virulence genes and clustered regularly interspaced short palindromic repeats (CRISPRs), we previously developed a sequence-based subtyping approach, designated CRISPR–multi-virulence-locus sequence typing (CRISPR-MVLST). To demonstrate the applicability of this approach, we analyzed a broad set of S. Newport isolates collected over a 5-year period by using CRISPR-MVLST and pulsed-field gel electrophoresis (PFGE). Among 84 isolates, we defined 38 S. Newport sequence types (NSTs), all of which were novel compared to our previous analyses, and 62 different PFGE patterns. Our data suggest that both subtyping approaches have high discriminatory abilities (>0.95) with a potential for clustering cases with common exposures. Importantly, we found that isolates from closely related NSTs were often similar by PFGE profile as well, further corroborating the applicability of CRISPR-MVLST. In the first full application of CRISPR-MVLST, we analyzed isolates from a recent S. Newport outbreak. In this blinded study, we confirmed the utility of CRISPR-MVLST and were able to distinguish the 10 outbreak isolates, as defined by PFGE and epidemiological data, from a collection of 20 S. Newport isolates. Together, our data show that CRISPR-MVLST could be a complementary approach to PFGE subtyping for S. Newport.

Antibiotic Resistance in Salmonella enterica Serovar Typhimurium Associates with CRISPR Sequence Type

ABSTRACT Salmonella enterica subsp. enterica serovar Typhimurium is a leading cause of food-borne salmonellosis in the United States. The number of antibiotic-resistant isolates identified in humans is steadily increasing, suggesting that the spread of antibiotic-resistant strains is a major threat to public health. S. Typhimurium is commonly identified in a wide range of animal hosts, food sources, and environments, but little is known about the factors mediating the spread of antibiotic resistance in this ecologically complex serovar. Previously, we developed a subtyping method, CRISPR–multi-virulence-locus sequence typing (MVLST), which discriminates among strains of several common S. enterica serovars. Here, CRISPR-MVLST identified 22 sequence types within a collection of 76 S. Typhimurium isolates from a variety of animal sources throughout central Pennsylvania. Six of the sequence types were identified in more than one isolate, and we observed statistically significant differences in resistance among these sequence types to 7 antibiotics commonly used in veterinary and human medicine, such as ceftiofur and ampicillin (P < 0.05). Importantly, five of these sequence types were subsequently identified in human clinical isolates, and a subset of these isolates had identical antibiotic resistance patterns, suggesting that these subpopulations are being transmitted through the food system. Therefore, CRISPR-MVLST is a promising subtyping method for monitoring the farm-to-fork spread of antibiotic resistance in S. Typhimurium.

Characterization and Evolution of Salmonella CRISPR-Cas Systems

Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) systems provide adaptive immunity from invasive genetic elements and encompass three essential features: (i) cas genes, (ii) a CRISPR array composed of spacers and direct repeats and (iii) an AT-rich leader sequence upstream of the array. We performed in-depth sequence analysis of the CRISPR-Cas systems in >600 Salmonella, representing four clinically prevalent serovars. Each CRISPR-Cas feature is extremely conserved in the Salmonella, and the CRISPR1 locus is more highly conserved than CRISPR2. Array composition is serovar-specific, although no convincing evidence of recent spacer acquisition against exogenous nucleic acids exists. Only 12% of spacers match phage and plasmid sequences and self-targeting spacers are associated with direct repeat variants. High nucleotide identity (>99.9%) exists across the cas operon among isolates of a single serovar and in some cases this conservation extends across divergent serovars. These observations reflect historical CRISPR-Cas immune activity, showing that this locus has ceased undergoing adaptive events. Intriguingly, the high level of conservation across divergent serovars shows that the genetic integrity of these inactive loci is maintained over time, contrasting with the canonical view that inactive CRISPR loci degenerate over time. This thorough characterization of Salmonella CRISPR-Cas systems presents new insights into Salmonella CRISPR evolution, particularly with respect to cas gene conservation, leader sequences, organization of direct repeats and protospacer matches. Collectively, our data suggest that Salmonella CRISPR-Cas systems are no longer immunogenic; rather, their impressive conservation indicates they may have an alternative function in Salmonella.Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) systems provide adaptive immunity from invasive genetic elements and encompass three essential features: (i) cas genes, (ii) a CRISPR array composed of spacers and direct repeats and (iii) an AT-rich leader sequence upstream of the array. We performed in-depth sequence analysis of the CRISPR-Cas systems in >600 Salmonella, representing four clinically prevalent serovars. Each CRISPR-Cas feature is extremely conserved in the Salmonella, and the CRISPR1 locus is more highly conserved than CRISPR2. Array composition is serovar-specific, although no convincing evidence of recent spacer acquisition against exogenous nucleic acids exists. Only 12 % of spacers match phage and plasmid sequences and self-targeting spacers are associated with direct repeat variants. High nucleotide identity (>99.9 %) exists across the cas operon among isolates of a single serovar and in some cases this conservation extends across divergent serovars. These observations reflect historical CRISPR-Cas immune activity, showing that this locus has ceased undergoing adaptive events. Intriguingly, the high level of conservation across divergent serovars shows that the genetic integrity of these inactive loci is maintained over time, contrasting with the canonical view that inactive CRISPR loci degenerate over time. This thorough characterization of Salmonella CRISPR-Cas systems presents new insights into Salmonella CRISPR evolution, particularly with respect to cas gene conservation, leader sequences, organization of direct repeats and protospacer matches. Collectively, our data suggest that Salmonella CRISPR-Cas systems are no longer immunogenic; rather, their impressive conservation indicates they may have an alternative function in Salmonella.

Prevalence and Spatial Distribution of Salmonella Infections in the Pennsylvania Raccoon (Procyon lotor)

A study was conducted to determine the prevalence and spatial distribution of Salmonella infection in Pennsylvania raccoons (Procyon lotor), common wildlife mammals known to occupy overlapping habitats with humans and domestic food animals. The Pennsylvania Game Commission provided a total of 371 raccoon intestinal samples from trapped and road‐killed raccoons collected between May and November 2011. Salmonella was isolated from the faeces of 56 (15.1%) of 371 raccoons in 35 (54%) of 65 counties across Pennsylvania. The five most frequently isolated serotypes were Newport (28.6%), Enteritidis (19.6%), Typhimurium (10.7%), Braenderup (8.9%) and Bareilly (7.1%). Pulsed‐field gel electrophoresis (PFGE) analysis of the Salmonella isolates and subsequent comparison to the Pennsylvania Department of Health human Salmonella PFGE database revealed 16 different pulsetypes in Salmonella isolates recovered from raccoons that were indistinguishable from pulsetypes of Salmonella collected from clinically ill humans during the study period. The pulsetypes of seven raccoon Salmonella isolates matched those of 56 human Salmonella isolates by month and geographical region of sample collection. Results from Clustered Regularly Interspaced Short Palindromic Repeats and Multi‐Virulence Locus Sequence Typing (CRISPR‐MVLST) analysis corroborated the PFGE and serotyping data. The findings of this study show that several PFGE pulsetypes of Salmonella were shared between humans and raccoons in Pennsylvania, indicating that raccoons and humans might share the same source of Salmonella.