Beth Tolar

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.

Genomic epidemiology of Salmonella enterica serotype Enteritidis based on population structure of prevalent lineages.

Major lineages emerged during the 17th–18th centuries and diversified during the 1920s and 1950s.

National Outbreak of Multidrug Resistant Salmonella Heidelberg Infections Linked to a Single Poultry Company.

Importance This large outbreak of foodborne salmonellosis demonstrated the complexity of investigating outbreaks linked to poultry products. The outbreak also highlighted the importance of efforts to strengthen food safety policies related to Salmonella in chicken parts and has implications for future changes within the poultry industry. Objective To investigate a large multistate outbreak of multidrug resistant Salmonella Heidelberg infections. Design Epidemiologic and laboratory investigations of patients infected with the outbreak strains of Salmonella Heidelberg and traceback of possible food exposures. Setting United States. Outbreak period was March 1, 2013 through July 11, 2014 Patients A case was defined as illness in a person infected with a laboratory-confirmed Salmonella Heidelberg with 1 of 7 outbreak pulsed-field gel electrophoresis (PFGE) XbaI patterns with illness onset from March 1, 2013 through July 11, 2014. A total of 634 case-patients were identified through passive surveillance; 200/528 (38%) were hospitalized, none died. Results Interviews were conducted with 435 case-patients: 371 (85%) reported eating any chicken in the 7 days before becoming ill. Of 273 case-patients interviewed with a focused questionnaire, 201 (74%) reported eating chicken prepared at home. Among case-patients with available brand information, 152 (87%) of 175 patients reported consuming Company A brand chicken. Antimicrobial susceptibility testing was completed on 69 clinical isolates collected from case-patients; 67% were drug resistant, including 24 isolates (35%) that were multidrug resistant. The source of Company A brand chicken consumed by case-patients was traced back to 3 California production establishments from which 6 of 7 outbreak strains were isolated. Conclusions Epidemiologic, laboratory, traceback, and environmental investigations conducted by local, state, and federal public health and regulatory officials indicated that consumption of Company A chicken was the cause of this outbreak. The outbreak involved multiple PFGE patterns, a variety of chicken products, and 3 production establishments, suggesting a reservoir for contamination upstream from the production establishments. Sources of bacteria and genes responsible for resistance, such as farms providing birds for slaughter or environmental reservoir on farms that raise chickens, might explain how multiple PFGE patterns were linked to chicken from 3 separate production establishments and many different poultry products.

Outbreaks of Salmonellosis From Small Turtles

OBJECTIVE: Turtle-associated salmonellosis (TAS), especially in children, is a reemerging public health issue. In 1975, small pet turtles (shell length <4 inches) sales were banned by federal law; reductions in pediatric TAS followed. Since 2006, the number of multistate TAS outbreaks has increased. We describe 8 multistate outbreaks with illness-onset dates occurring in 2011–2013. METHODS: We conducted epidemiologic, environmental, and traceback investigations. Cases were defined as infection with ≥1 of 10 molecular subtypes of Salmonella Sandiego, Pomona, Poona, Typhimurium, and I 4,[5],12:i:-. Water samples from turtle habitats linked to human illnesses were cultured for Salmonella. RESULTS: We identified 8 outbreaks totaling 473 cases from 41 states, Washington DC, and Puerto Rico with illness onsets during May 2011–September 2013. The median patient age was 4 years (range: 1 month–94 years); 45% percent were Hispanic; and 28% were hospitalized. In the week preceding illness, 68% (187 of 273) of case-patients reported turtle exposure; among these, 88% (124 of 141) described small turtles. Outbreak strains were isolated from turtle habitats linked to human illnesses in seven outbreaks. Traceback investigations identified 2 Louisiana turtle farms as the source of small turtles linked to 1 outbreak; 1 outbreak strain was isolated from turtle pond water from 1 turtle farm. CONCLUSIONS: Eight multistate outbreaks associated with small turtles were investigated during 2011–2013. Children <5 years and Hispanics were disproportionately affected. Prevention efforts should focus on patient education targeting families with young children and Hispanics and enactment of state and local regulations to complement federal sales restrictions.

Characterization of blaCMY Plasmids and Their Possible Role in Source Attribution of Salmonella enterica Serotype Typhimurium Infections

Salmonella is an important cause of foodborne illness; however, identifying the source of these infections can be difficult. This is especially true for Salmonella serotype Typhimurium, which is found in diverse agricultural niches. Extended-spectrum cephalosporins (ESC) are one of the primary treatment choices for complicated Salmonella infections. In Salmonella, ESC resistance in the United States is mainly mediated by blaCMY genes carried on various plasmids. In this study, we examined whether the characterization of blaCMY plasmids, along with additional information, can help us identify potential sources of infection by Salmonella, and used serotype Typhimurium as a model. In the United States, monitoring of retail meat, food animals, and ill persons for antimicrobial-resistant Salmonella is conducted by the National Antimicrobial Resistance Monitoring System. In 2008, 70 isolates (70/581; 12.0%) (34 isolates from retail meat, 23 food animal, and 13 human) were resistant to ceftriaxone and amoxicillin/clavulanic acid. All were polymerase chain reaction (PCR)-positive for blaCMY and 59/70 (84.3%) of these genes were plasmid encoded. PCR-based replicon typing identified 42/59 (71.2%) IncI1-blaCMY plasmids and 17/59 (28.8%) IncA/C-blaCMY plasmids. Isolates from chickens or chicken products with blaCMY plasmids primarily had IncI1-blaCMY plasmids (37/40; 92.5%), while all isolates from cattle had IncA/C-blaCMY plasmids. Isolates from humans had either IncA/C- blaCMY (n=8/12; [66.7%]) or IncI1- blaCMY (n=4/12 [33.3%]) plasmids. All of the IncI1-blaCMY plasmids were ST12 or were closely related to ST12. Antimicrobial susceptibility patterns (AST) and pulsed-field gel electrophoresis (PFGE) patterns of the isolates were also compared and differences were identified between isolate sources. When the source of a Typhimurium outbreak or sporadic illness is unknown, characterizing the outbreak isolates blaCMY plasmids, AST, and PFGE patterns may help identify it.

Identification and Characterization of Multidrug-Resistant Salmonella enterica Serotype Albert Isolates in the United States

ABSTRACT Salmonella enterica is one of the most common causes of bacterial foodborne illness in the United States. Although most Salmonella infections are self-limiting, antimicrobial treatment of invasive salmonellosis is critical. The primary antimicrobial treatment options include fluoroquinolones or extended-spectrum cephalosporins, and resistance to these antimicrobial drugs may complicate treatment. At present, S. enterica is composed of more than 2,600 unique serotypes, which vary greatly in geographic prevalence, ecological niche, and the ability to cause human disease, and it is important to understand and mitigate the source of human infection, particularly when antimicrobial resistance is found. In this study, we identified and characterized 19 S. enterica serotype Albert isolates collected from food animals, retail meat, and humans in the United States during 2005 to 2013. All five isolates from nonhuman sources were obtained from turkeys or ground turkey, and epidemiologic data suggest poultry consumption or live-poultry exposure as the probable source of infection. S. enterica serotype Albert also appears to be geographically localized to the midwestern United States. All 19 isolates displayed multidrug resistance, including decreased susceptibility to fluoroquinolones and resistance to extended-spectrum cephalosporins. Turkeys are a likely source of multidrug-resistant S. enterica serotype Albert, and circulation of resistance plasmids, as opposed to the expansion of a single resistant strain, is playing a role. More work is needed to understand why these resistance plasmids spread and how their presence and the serotype they reside in contribute to human disease.

Outbreak of Salmonella Oslo Infections Linked to Persian Cucumbers - United States, 2016.

In April 2016, PulseNet, the national molecular subtyping network for foodborne disease surveillance, detected a multistate cluster of Salmonella enterica serotype Oslo infections with an indistinguishable pulsed-field gel electrophoresis (PFGE) pattern (XbaI PFGE pattern OSLX01.0090).* This PFGE pattern was new in the database; no previous infections or outbreaks have been identified. CDC, state and local health and agriculture departments and laboratories, and the Food and Drug Administration (FDA) conducted epidemiologic, traceback, and laboratory investigations to identify the source of this outbreak. A total of 14 patients in eight states were identified, with illness onsets occurring during March 21-April 9, 2016. Whole genome sequencing, a highly discriminating subtyping method, was used to further characterize PFGE pattern OSLX01.0090 isolates. Epidemiologic evidence indicates Persian cucumbers as the source of Salmonella Oslo infections in this outbreak. This is the fourth identified multistate outbreak of salmonellosis associated with cucumbers since 2013. Further research is needed to understand the mechanism and factors that contribute to contamination of cucumbers during growth, harvesting, and processing to prevent future outbreaks.

Epidemiology of Salmonellaenterica Serotype Dublin Infections among Humans, United States, 1968–2013

Infection incidence and antimicrobial drug resistance are increasing.

Utility of Combining Whole Genome Sequencing with Traditional Investigational Methods To Solve Foodborne Outbreaks of Salmonella Infections Associated with Chicken: A New Tool for Tackling This Challenging Food Vehicle

High consumption rates and a multitude of brands make multistate foodborne outbreaks of Salmonella infections associated with chicken challenging to investigate, but whole genome sequencing is a powerful tool that can be used to assist investigators. Whole genome sequencing of pathogens isolated from clinical, environmental, and food samples is increasingly being used in multistate foodborne outbreak investigations to determine with unprecedented resolution how closely related these isolates are to one another genetically. In 2014, federal and state health officials investigated an outbreak of 146 Salmonella Heidelberg infections in 24 states. A follow-up analysis was conducted after the conclusion of the investigation in which 27 clinical and 24 food isolates from the outbreak underwent whole genome sequencing. These isolates formed seven clades, the largest of which contained clinical isolates from a subcluster of case patients who attended a catered party. One isolate from a chicken processed by a large producer was closely related genetically (zero to three single-nucleotide polymorphism differences) to the clinical isolates from these subcluster case patients. Chicken from this large producer was also present in the kitchen of the caterer on the day before the event, thus providing additional evidence that the chicken from this producer was the outbreak source. This investigation highlights how whole genome sequencing can be used with epidemiologic and traceback evidence to identify chicken sources of foodborne outbreaks.