Recurrent de novo SPTLC2 variant causes childhood-onset amyotrophic lateral sclerosis (ALS) by excess sphingolipid synthesis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of the upper and lower motor neurons with varying ages of onset, progression and pathomechanisms. Monogenic childhood-onset ALS, although rare, forms an important subgroup of ALS. We recently reported specific SPTLC1 variants resulting in sphingolipid overproduction as a cause for juvenile ALS. Here, we report six patients from six independent families with a recurrent, de novo, heterozygous variant in SPTLC2 c.778G>A [p.Glu260Lys] manifesting with juvenile ALS. METHODS: Clinical examination of the patients along with ancillary and genetic testing, followed by biochemical investigation of patients' blood and fibroblasts, was performed. RESULTS: All patients presented with early-childhood-onset progressive weakness, with signs and symptoms of upper and lower motor neuron degeneration in multiple myotomes, without sensory neuropathy. These findings were supported on ancillary testing including nerve conduction studies and electromyography, muscle biopsies and muscle ultrasound studies. Biochemical investigations in plasma and fibroblasts showed elevated levels of ceramides and unrestrained de novo sphingolipid synthesis. Our studies indicate that SPTLC2 variant [c.778G>A, p.Glu260Lys] acts distinctly from hereditary sensory and autonomic neuropathy (HSAN)-causing SPTLC2 variants by causing excess canonical sphingolipid biosynthesis, similar to the recently reported SPTLC1 ALS associated pathogenic variants. Our studies also indicate that serine supplementation, which is a therapeutic in SPTLC1 and SPTCL2-associated HSAN, is expected to exacerbate the excess sphingolipid synthesis in serine palmitoyltransferase (SPT)-associated ALS. CONCLUSIONS: SPTLC2 is the second SPT-associated gene that underlies monogenic, juvenile ALS and further establishes alterations of sphingolipid metabolism in motor neuron disease pathogenesis. Our findings also have important therapeutic implications: serine supplementation must be avoided in SPT-associated ALS, as it is expected to drive pathogenesis further.

Epidemiology of Pulmonary and Extrapulmonary Nontuberculous Mycobacteria Infections at 4 US Emerging Infections Program Sites: A 6-Month Pilot.

BACKGROUND: Nontuberculous mycobacteria (NTM) cause pulmonary (PNTM) and extrapulmonary (ENTM) disease. Infections are difficult to diagnose and treat, and exposures occur in healthcare and community settings. In the United States, NTM epidemiology has been described largely through analyses of microbiology data from health departments, electronic health records, and administrative data. We describe findings from a multisite pilot of active, laboratory- and population-based NTM surveillance. METHODS: The Centers for Disease Control and Prevention's Emerging Infections Program conducted NTM surveillance at 4 sites (Colorado, 5 counties; Minnesota, 2 counties; New York, 2 counties; and Oregon, 3 counties [PNTM] and statewide [ENTM]) from 1 October 2019 through 31 March 2020. PNTM cases were defined using published microbiologic criteria. ENTM cases required NTM isolation from a nonpulmonary specimen, excluding stool and rectal swabs. Patient data were collected via medical record review. RESULTS: Overall, 299 NTM cases were reported (PNTM: 231, 77%); Mycobacterium avium complex was the most common species group. Annualized prevalence was 7.5/100 000 population (PNTM: 6.1/100 000; ENTM: 1.4/100 000). Most patients had signs or symptoms in the 14 days before positive specimen collection (ENTM: 62, 91.2%; PNTM: 201, 87.0%). Of PNTM cases, 145 (62.8%) were female and 168 (72.7%) had underlying chronic lung disease. Among ENTM cases, 29 (42.6%) were female, 21 (30.9%) did not have documented underlying conditions, and 26 (38.2%) had infection at the site of a medical device or procedure. CONCLUSIONS: Active, population-based NTM surveillance will provide data for monitoring the burden of disease and characterize affected populations to inform interventions.

Listeria monocytogenes Illness and Deaths Associated With Ongoing Contamination of a Multiregional Brand of Ice Cream Products, United States, 2010-2015.

BACKGROUND: Frozen foods have rarely been linked to Listeria monocytogenes illness. We describe an outbreak investigation prompted by both hospital clustering of illnesses and product testing. METHODS: We identified outbreak-associated listeriosis cases using whole-genome sequencing (WGS), product testing results, and epidemiologic linkage to cases in the same Kansas hospital. We reviewed hospital medical and dietary records, product invoices, and molecular subtyping results. Federal and state officials tested product and environmental samples for L. monocytogenes. RESULTS: Kansas officials were investigating 5 cases of listeriosis at a single hospital when, simultaneously, unrelated sampling for a study in South Carolina identified L. monocytogenes in Company A ice cream products made in Texas. Isolates from 4 patients and Company A products were closely related by WGS, and the 4 patients with known exposures had consumed milkshakes made with Company A ice cream while hospitalized. Further testing identified L. monocytogenes in ice cream produced in a second Company A production facility in Oklahoma; these isolates were closely related by WGS to those from 5 patients in 3 other states. These 10 illnesses, involving 3 deaths, occurred from 2010 through 2015. Company A ultimately recalled all products. CONCLUSIONS: In this US outbreak of listeriosis linked to a widely distributed brand of ice cream, WGS and product sampling helped link cases spanning 5 years to 2 production facilities, indicating longstanding contamination. Comprehensive sanitation controls and environmental and product testing for L. monocytogenes with regulatory oversight should be implemented for ice cream production.

Case Series of Thrombosis With Thrombocytopenia Syndrome After COVID-19 Vaccination-United States, December 2020 to August 2021.

BACKGROUND: Thrombosis with thrombocytopenia syndrome (TTS) is a potentially life-threatening condition associated with adenoviral-vectored COVID-19 vaccination. It presents similarly to spontaneous heparin-induced thrombocytopenia. Twelve cases of cerebral venous sinus thrombosis after vaccination with the Ad26.COV2.S COVID-19 vaccine (Janssen/Johnson & Johnson) have previously been described. OBJECTIVE: To describe surveillance data and reporting rates of all reported TTS cases after COVID-19 vaccination in the United States. DESIGN: Case series. SETTING: United States. PATIENTS: Case patients receiving a COVID-19 vaccine from 14 December 2020 through 31 August 2021 with thrombocytopenia and thrombosis (excluding isolated ischemic stroke or myocardial infarction) reported to the Vaccine Adverse Event Reporting System. If thrombosis was only in an extremity vein or pulmonary embolism, a positive enzyme-linked immunosorbent assay for antiplatelet factor 4 antibodies or functional heparin-induced thrombocytopenia platelet test result was required. MEASUREMENTS: Reporting rates (cases per million vaccine doses) and descriptive epidemiology. RESULTS: A total of 57 TTS cases were confirmed after vaccination with Ad26.COV2.S (n = 54) or a messenger RNA (mRNA)-based COVID-19 vaccine (n = 3). Reporting rates for TTS were 3.83 per million vaccine doses (Ad26.COV2.S) and 0.00855 per million vaccine doses (mRNA-based COVID-19 vaccines). The median age of patients with TTS after Ad26.COV2.S vaccination was 44.5 years (range, 18 to 70 years), and 69% of patients were women. Of the TTS cases after mRNA-based COVID-19 vaccination, 2 occurred in men older than 50 years and 1 in a woman aged 50 to 59 years. All cases after Ad26.COV2.S vaccination involved hospitalization, including 36 (67%) with intensive care unit admission. Outcomes of hospitalizations after Ad26.COV2.S vaccination included death (15%), discharge to postacute care (17%), and discharge home (68%). LIMITATIONS: Underreporting and incomplete case follow-up. CONCLUSION: Thrombosis with thrombocytopenia syndrome is a rare but serious adverse event associated with Ad26.COV2.S vaccination. The different demographic characteristics of the 3 cases reported after mRNA-based COVID-19 vaccines and the much lower reporting rate suggest that these cases represent a background rate. PRIMARY FUNDING SOURCE: Centers for Disease Control and Prevention.

Genome sequencing as a first-line diagnostic test for hospitalized infants.

PURPOSE: SouthSeq is a translational research study that undertook genome sequencing (GS) for infants with symptoms suggestive of a genetic disorder. Recruitment targeted racial/ethnic minorities and rural, medically underserved areas in the Southeastern United States, which are historically underrepresented in genomic medicine research. METHODS: GS and analysis were performed for 367 infants to detect disease-causal variation concurrent with standard of care evaluation and testing. RESULTS: Definitive diagnostic (DD) or likely diagnostic (LD) genetic findings were identified in 30% of infants, and 14% of infants harbored an uncertain result. Only 43% of DD/LD findings were identified via concurrent clinical genetic testing, suggesting that GS testing is better for obtaining early genetic diagnosis. We also identified phenotypes that correlate with the likelihood of receiving a DD/LD finding, such as craniofacial, ophthalmologic, auditory, skin, and hair abnormalities. We did not observe any differences in diagnostic rates between racial/ethnic groups. CONCLUSION: We describe one of the largest-to-date GS cohorts of ill infants, enriched for African American and rural patients. Our results show the utility of GS because it provides early-in-life detection of clinically relevant genetic variations not detected by current clinical genetic testing, particularly for infants exhibiting certain phenotypic features.

Newborn screening for Pompe disease: Parental experiences and follow-up care for a late-onset diagnosis.

Newborn screening (NBS) for Pompe disease (PD) was added to the Recommended Uniform Screening Panel (RUSP) in the United States in 2015 because there was compelling evidence of health benefits for early diagnosis of Infantile-onset Pompe disease (IOPD). However, one limitation of NBS for PD is its inability to distinguish IOPD and late onset forms of Pompe disease (LOPD). Management of LOPD is challenging because of uncertainty around progression of LOPD and determining the appropriate time for treatment initiation. The aims of this study were to understand the impact of LOPD identified through NBS, by exploring the differences in attitudes, emotions and opinions among parents and identify their needs for follow-up care. Study participants were recruited from states that included PD on their NBS panel. Semi-structured interviews were conducted with parents of nine children who were diagnosed with LOPD after an abnormal NBS result. Predominantly, parents reported a lack of adequate information, guidance, and psychosocial support from the very beginning and through the course of their diagnosis. This caused uncertainty, anxiety, frustration, and fear of the unknown. Parents live in a 'worry or not to worry' phase, balancing between coping methods to avoid over medicalization of their child, but also preparing concrete follow-up plans to be on the lookout for any signs of PD-related symptoms. Understanding parents' experiences allows genetic counselors and NBS programs to proactively design care plan for parents during this difficult period.

Perceived monoracism and psychological adjustment of multiracial adults: The roles of racially diverse contexts and creating third space.

Across two studies, we examined whether racially diverse contexts in combination with creating a third (multiracial) space played a protective role in the association between perceived monoracism and psychological adjustment for multiracial adults. Study 1 participants (N = 263; 77.8% female, Mage = 32.16 years) were recruited from national multiracial organizations and completed an online cross-sectional survey in 2009. Study 2 participants (N = 1478; 56.2% female, Mage = 48.89 years) were recruited by the Pew Research Center and completed a nationally representative survey in 2015. Results indicated significant three-way interaction effects on psychological adjustment, with differential effects depending on the types of perceived monoracism. Specifically, when subjective racial diversity was high, creating a third space buffered against the deleterious effects of multiracial discrimination on distress, negative affect, and life satisfaction; while it exacerbated the link between perceived racial ambiguity and negative affect. This study provides empirical evidence for the importance of using an ecological framework when examining multiracial identity and experiences. Results also highlight the need to move beyond conceptualizing and measuring multiracial experiences as dichotomous risks or resiliencies. Rather, we should aim to understand multiracial experiences as either promoting or inhibiting across contexts, time, and individual characteristics.

Public Health Importance of Invasive Methicillin-sensitive Staphylococcus aureus Infections: Surveillance in 8 US Counties, 2016.

BACKGROUND: Public health and infection control prevention and surveillance efforts in the United States have primarily focused on methicillin-resistant Staphylococcus aureus (MRSA). We describe the public health importance of methicillin-susceptible S. aureus (MSSA) in selected communities. METHODS: We analyzed Emerging Infections Program surveillance data for invasive S. aureus (SA) infections (isolated from a normally sterile body site) in 8 counties in 5 states during 2016. Cases were considered healthcare-associated if culture was obtained >3 days after hospital admission; if associated with dialysis, hospitalization, surgery, or long-term care facility (LTCF) residence within 1 year prior; or if a central venous catheter was present ≤2 days prior. Incidence per 100 000 census population was calculated, and a multivariate logistic regression model with random intercepts was used to compare MSSA risk factors with those of MRSA. RESULTS: Invasive MSSA incidence (31.3/100 000) was 1.8 times higher than MRSA (17.5/100 000). Persons with MSSA were more likely than those with MRSA to have no underlying medical conditions (adjusted odds ratio [aOR], 2.06; 95% confidence interval [CI], 1.26-3.39) and less likely to have prior hospitalization (aOR, 0.70; 95% CI, 0.60-0.82) or LTCF residence (aOR, 0.37; 95% CI, 0.29-0.47). MSSA accounted for 59.7% of healthcare-associated cases and 60.1% of deaths. CONCLUSIONS: Although MRSA tended to be more closely associated with healthcare exposures, invasive MSSA is a substantial public health problem in the areas studied. Public health and infection control prevention efforts should consider MSSA prevention in addition to MRSA.

Multistate Outbreak of Listeriosis Associated with Packaged Leafy Green Salads, United States and Canada, 2015-2016.

We investigated an outbreak of listeriosis detected by whole-genome multilocus sequence typing and associated with packaged leafy green salads. Nineteen cases were identified in the United States during July 5, 2015-January 31, 2016; isolates from case-patients were closely related (median difference 3 alleles, range 0-16 alleles). Of 16 case-patients interviewed, all reported salad consumption. Of 9 case-patients who recalled brand information, all reported brands processed at a common US facility. The Public Health Agency of Canada simultaneously investigated 14 cases of listeriosis associated with this outbreak. Isolates from the processing facility, packaged leafy green salads, and 9 case-patients from Canada were closely related to US clinical isolates (median difference 3 alleles, range 0-16 alleles). This investigation led to a recall of packaged leafy green salads made at the processing facility. Additional research is needed to identify best practices and effective policies to reduce the likelihood of Listeria monocytogenes contamination of fresh produce.

Bacterial and Fungal Infections in Persons Who Inject Drugs - Western New York, 2017.

During 2014-2017, CDC Emerging Infections Program surveillance data reported that the occurrence of invasive methicillin-resistant Staphylococcus aureus (MRSA) infections associated with injection drug use doubled among persons aged 18-49 years residing in Monroe County in western New York.* Unpublished surveillance data also indicate that an increasing proportion of all Candida spp. bloodstream infections in Monroe County and invasive group A Streptococcus (GAS) infections in 15 New York counties are also occurring among persons who inject drugs. In addition, across six surveillance sites nationwide, the proportion of invasive MRSA infections that occurred in persons who inject drugs increased from 4.1% of invasive MRSA cases in 2011 to 9.2% in 2016 (1). To better understand the types and frequency of these infections and identify prevention opportunities, CDC and public health partners conducted a rapid assessment of bacterial and fungal infections among persons who inject drugs in western New York. The goals were to assess which bacterial and fungal pathogens most often cause infections in persons who inject drugs, what proportion of persons who inject use opioids, and of these, how many were offered medication-assisted treatment for opioid use disorder. Medication-assisted treatment, which includes use of medications such as buprenorphine, methadone, and naltrexone, reduces cravings and has been reported to lower the risk for overdose death and all-cause mortality in persons who use opioids (2,3). In this assessment, nearly all persons with infections who injected drugs used opioids (97%), but half of inpatients (22 of 44) and 12 of 13 patients seen only in the emergency department (ED) were not offered medication-assisted treatment. The most commonly identified pathogen was S. aureus (80%), which is frequently found on skin. Health care visits for bacterial and fungal infections associated with injection opioid use are an opportunity to treat the underlying opioid use disorder with medication-assisted treatment. Routine care for patients who continue to inject should include advice on hand hygiene and not injecting into skin that has not been cleaned or to use any equipment contaminated by reuse, saliva, soil, or water (4,5).

Listeriosis Outbreaks Associated with Soft Cheeses, United States, 1998-20141.

Since 2006, the number of reported US listeriosis outbreaks associated with cheese made under unsanitary conditions has increased. Two-thirds were linked to Latin-style soft cheese, often affecting pregnant Hispanic women and their newborns. Adherence to pasteurization protocols and sanitation measures to avoid contamination after pasteurization can reduce future outbreaks.

Heritable variation in prey defence provides refuge for subdominant predators.

Generalist predators with broadly overlapping niches commonly coexist on seemingly identical sets of prey. Here, we provide empirical demonstration that predators can differentially exploit fine-grained niches generated by variable, heritable and selective defences within a single prey species. Some, but not all, clones of the aphid Aphis craccivora are toxic towards the dominant invasive predatory ladybeetle, Harmonia axyridis However, other less competitive ladybeetle species are not affected by the aphid's toxic trait. In laboratory and open field experiments, we show: (i) that subdominant ladybeetle species were able to exploit the toxic aphids, benefitting from the suppression of the dominant predator; and (ii) that this narrow-spectrum toxicity can function as an anti-predator defence for the aphid, but depends on enemy community context. Our results demonstrate that niche differentiation among generalist predators may hinge upon previously underappreciated heritable variation in prey defence, which, in turn, may promote diversity and stability of enemy communities invaded by a dominant predator.

Invasive Methicillin-Resistant Staphylococcus aureus Infections Among Persons Who Inject Drugs - Six Sites, 2005-2016.

In the United States, age-adjusted opioid overdose death rates increased by >200% during 1999-2015, and heroin overdose death rates increased nearly 300% during 2011-2015 (1). During 2011-2013, the rate of heroin use within the past year among U.S. residents aged ≥12 years increased 62.5% overall and 114.3% among non-Hispanic whites, compared with 2002-2004 (2). Increases in human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections related to increases in injection drug use have been recently highlighted (3,4); likewise, invasive bacterial infections, including endocarditis, osteomyelitis, and skin and soft tissue infections, have increased in areas where the opioid epidemic is expanding (5-7). To assess the effects of the opioid epidemic on invasive methicillin-resistant Staphylococcus aureus (MRSA) infections during 2005-2016, surveillance data from CDC's Emerging Infections Program (EIP) were analyzed (8). Persons who inject drugs were estimated to be 16.3 times more likely to develop invasive MRSA infections than others. The proportion of invasive MRSA cases that occurred among persons who inject drugs increased from 4.1% in 2011 to 9.2% in 2016. Infection types were frequently those associated with nonsterile injection drug use. Continued increases in nonsterile injection drug use are likely to result in increases in invasive MRSA infections, underscoring the importance of public health measures to curb the opioid epidemic.

Clinical Characteristics and Ancillary Test Results Among Patients With Botulism-United States, 2002-2015.

Background: Botulism is classically described as a bilateral, symmetric, descending flaccid paralysis in an afebrile and alert patient without sensory findings. We describe the reported spectrum of clinical findings among persons >12 months of age in the United States during 2002-2015. Methods: The Centers for Disease Control and Prevention collects clinical findings reported by physicians treating suspected cases of botulism nationwide. We analyzed symptoms and signs, and neuroimaging and cerebrospinal fluid (CSF) results. A case was defined as illness compatible with botulism with laboratory confirmation or epidemiologic link to a confirmed case, and presence or absence of at least 1 sign or symptom recorded. Physicians' differential diagnoses were evaluated. Results: Clinical information was evaluated for 332 botulism cases; data quality and completeness were variable. Most had no fever (99%), descending paralysis (93%), no mental status change (91%), at least 1 ocular weakness finding (84%), and neuroimaging without acute changes (82%). Some had paresthesias (17%), elevated CSF protein level (13%), and other features sometimes considered indicative of alternative diagnoses. Five of 71 (7%) cases with sufficient information were reported to have atypical findings (eg, at least 1 cranial nerve finding that was unilateral or ascending paralysis). Illnesses on the physician differential included Guillain-Barré syndrome (99 cases) and myasthenia gravis (76 cases) and, rarely, gastrointestinal-related illness (5 cases), multiple sclerosis (3 cases), sepsis (3 cases), and Lyme disease (2 cases). Conclusions: Our analysis illustrates that classic symptoms and signs were common among patients with botulism but that features considered atypical were reported by some physicians. Diagnosis can be challenging, as illustrated by the broad range of illnesses on physician differentials.

Assessment of the Incubation Period for Invasive Listeriosis.

We characterized incubation periods among outbreak-associated listeriosis cases, using a simulation model to account for patients with multiple exposure dates. The median was 11 days; 90% of cases occurred within 28 days, and incubation periods varied by clinical manifestation.

Implementation of Nationwide Real-time Whole-genome Sequencing to Enhance Listeriosis Outbreak Detection and Investigation.

Listeria monocytogenes (Lm) causes severe foodborne illness (listeriosis). Previous molecular subtyping methods, such as pulsed-field gel electrophoresis (PFGE), were critical in detecting outbreaks that led to food safety improvements and declining incidence, but PFGE provides limited genetic resolution. A multiagency collaboration began performing real-time, whole-genome sequencing (WGS) on all US Lm isolates from patients, food, and the environment in September 2013, posting sequencing data into a public repository. Compared with the year before the project began, WGS, combined with epidemiologic and product trace-back data, detected more listeriosis clusters and solved more outbreaks (2 outbreaks in pre-WGS year, 5 in WGS year 1, and 9 in year 2). Whole-genome multilocus sequence typing and single nucleotide polymorphism analyses provided equivalent phylogenetic relationships relevant to investigations; results were most useful when interpreted in context of epidemiological data. WGS has transformed listeriosis outbreak surveillance and is being implemented for other foodborne pathogens.

Multistate outbreak of listeriosis associated with cantaloupe.

BACKGROUND: Although new pathogen-vehicle combinations are increasingly being identified in produce-related disease outbreaks, fresh produce is a rarely recognized vehicle for listeriosis. We investigated a nationwide listeriosis outbreak that occurred in the United States during 2011. METHODS: We defined an outbreak-related case as a laboratory-confirmed infection with any of five outbreak-related subtypes of Listeria monocytogenes isolated during the period from August 1 through October 31, 2011. Multistate epidemiologic, trace-back, and environmental investigations were conducted, and outbreak-related cases were compared with sporadic cases reported previously to the Listeria Initiative, an enhanced surveillance system that routinely collects detailed information about U.S. cases of listeriosis. RESULTS: We identified 147 outbreak-related cases in 28 states. The majority of patients (127 of 147, 86%) were 60 years of age or older. Seven infections among pregnant women and newborns and one related miscarriage were reported. Of 145 patients for whom information about hospitalization was available, 143 (99%) were hospitalized. Thirty-three of the 147 patients (22%) died. Patients with outbreak-related illness were significantly more likely to have eaten cantaloupe than were patients 60 years of age or older with sporadic illness (odds ratio, 8.5; 95% confidence interval, 1.3 to ∞). Cantaloupe and environmental samples collected during the investigation yielded isolates matching all five outbreak-related subtypes, confirming that whole cantaloupe produced by a single Colorado farm was the outbreak source. Unsanitary conditions identified in the processing facility operated by the farm probably resulted in contamination of cantaloupes with L. monocytogenes. CONCLUSIONS: Raw produce, including cantaloupe, can serve as a vehicle for listeriosis. This outbreak highlights the importance of preventing produce contamination within farm and processing environments.

Two Listeria monocytogenes Pseudo-outbreaks Caused by Contaminated Laboratory Culture Media.

Listeriosis is a serious foodborne infection that disproportionately affects elderly adults, pregnant women, newborns, and immunocompromised individuals. Diagnosis is made by culturing Listeria monocytogenes from sterile body fluids or from products of conception. This report describes the investigations of two listeriosis pseudo-outbreaks caused by contaminated laboratory media made from sheep blood.

Determination of Evolutionary Relationships of Outbreak-Associated Listeria monocytogenes Strains of Serotypes 1/2a and 1/2b by Whole-Genome Sequencing.

We used whole-genome sequencing to determine evolutionary relationships among 20 outbreak-associated clinical isolates of Listeria monocytogenes serotypes 1/2a and 1/2b. Isolates from 6 of 11 outbreaks fell outside the clonal groups or "epidemic clones" that have been previously associated with outbreaks, suggesting that epidemic potential may be widespread in L. monocytogenes and is not limited to the recognized epidemic clones. Pairwise comparisons between epidemiologically related isolates within clonal complexes showed that genome-level variation differed by 2 orders of magnitude between different comparisons, and the distribution of point mutations (core versus accessory genome) also varied. In addition, genetic divergence between one closely related pair of isolates from a single outbreak was driven primarily by changes in phage regions. The evolutionary analysis showed that the changes could be attributed to horizontal gene transfer; members of the diverse bacterial community found in the production facility could have served as the source of novel genetic material at some point in the production chain. The results raise the question of how to best utilize information contained within the accessory genome in outbreak investigations. The full magnitude and complexity of genetic changes revealed by genome sequencing could not be discerned from traditional subtyping methods, and the results demonstrate the challenges of interpreting genetic variation among isolates recovered from a single outbreak. Epidemiological information remains critical for proper interpretation of nucleotide and structural diversity among isolates recovered during outbreaks and will remain so until we understand more about how various population histories influence genetic variation.