Food Safety and Inspection Service

Antimicrobial resistance (AMR) is a significant public health threat. With the rise of affordable whole genome sequencing, in silico approaches to assessing AMR gene content can be used to detect known resistance mechanisms and potentially identify novel mechanisms. To enable accurate assessment of AMR gene content, as part of a multi-agency collaboration, NCBI developed a comprehensive AMR gene database, the Bacterial Antimicrobial Resistance Reference Gene Database and the AMR gene detection tool AMRFinder. Here, we describe the expansion of the Reference Gene Database, now called the Reference Gene Catalog, to include putative acid, biocide, metal, stress resistance genes, in addition to virulence genes and species-specific point mutations. Genes and point mutations are classified by broad functions, as well as more detailed functions. As we have expanded both the functional repertoire of identified genes and functionality, NCBI released a new version of AMRFinder, known as AMRFinderPlus. This new tool allows users the option to utilize only the core set of AMR elements, or include stress response and virulence genes, too. AMRFinderPlus can detect acquired genes and point mutations in both protein and nucleotide sequence. In addition, the evidence used to identify the gene has been expanded to include whether nucleotide or protein sequence was used, its location in the contig, and presence of an internal stop codon. These database improvements and functional expansions will enable increased precision in identifying AMR genes, linking AMR genotypes and phenotypes, and determining possible relationships between AMR, virulence, and stress response.

isolates phenotypically tested against various antimicrobial agents. Of 87,679 susceptibility tests performed, 98.4% were consistent with predictions. To assess the accuracy of AMRFinder, we compared its gene symbol output with that of a 2017 version of ResFinder, another publicly available resistance gene detection system. Most gene calls were identical, but there were 1,229 gene symbol differences (8.8%) between them, with differences due to both algorithmic differences and database composition. AMRFinder missed 16 loci that ResFinder found, while ResFinder missed 216 loci that AMRFinder identified. Based on these results, AMRFinder appears to be a highly accurate AMR gene detection system.

There would appear to be little argument that the large outbreaks of E. coli O157:H7 which have occurred since the early 1980s represent a distinct, new phenomenon. The number of reported cases have increased dramatically, starting from zero in 1981; however, it is also clear that this increase in reported cases is in part an artifact of improved surveillance and reporting. Available data suggest that E. coli O157:H7 infections were present prior to 1982, although numbers appear to have been small. At a molecular level, the organism shows evidence of clonal origin, but there is not the striking clonality, with virtually identical pulsed-field gel electrophoresis and ribotyping patterns, which has been seen in situations such as the emergence of Vibrio cholerae O139 Bengal in the Indian subcontinent in 1992 or the introduction of V. cholerae O1 into naïve populations in South America in 1991 (127-129). Findings are more consistent with the image of an organism which arose from a common ancestor, but which has had time to become distributed geographically and to show some evidence of genetic divergence. While this is an "emerging" infection, at least in terms of its distribution and public recognition, it is unlikely that it will be possible to identify the "first" O157:H7 case or to track the clonal spread of the organism through cattle or human populations.

OBJECTIVES: This study evaluated the production of dry fermented salami associated with an outbreak of Escherichia coli O157.H7 infection in Washington State and California. METHODS: Facility inspections, review of plant monitoring data, food handler interviews, and microbiological testing of salami products were conducted. RESULTS: Production methods complied with federal requirements and industry-developed good manufacturing practices. No evidence suggested that postprocessing contamination occurred. Calculations suggested that the infectious dose was smaller than 50 E. coli O157:H7 bacteria. CONCLUSIONS: Dry fermented salami can serve as a vehicle of transmission for O157:H7 strains. Our investigation and prior laboratory studies suggest that E. coli O157:H7 can survive currently accepted processing methods.

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.

Poly (ADP-ribose) polymerase-1 (PARP1) is a key facilitator of DNA repair and is implicated in pathways of tumorigenesis. PARP inhibitors have gained recent attention as rationally designed therapeutics for the treatment of several malignancies, particularly those associated with dysfunctional DNA repair pathways, including triple-negative breast cancer (TNBC). We investigated the PARP1 gene expression profile in surgical samples from more than 8,000 primary malignant and normal human tissues. PARP1 expression was found to be significantly increased in several malignant tissues, including those isolated from patients with breast, uterine, lung, ovarian, and skin cancers, and non-Hodgkin's lymphoma. Within breast infiltrating ductal carcinoma (IDC) samples tested, mean PARP1 expression was significantly higher relative to normal breast tissue, with over 30% of IDC samples demonstrating upregulation of PARP1, compared with 2.9% of normal tissues. Because of known DNA repair defects, including BRCA1 dysfunction, associated with TNBC, exploration of PARP1 expression in breast cancers related to expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) led to the observation that negative expression of any of the 3 receptors was associated with upregulation of PARP1 expression, compared with receptor-positive tissues. To validate these observations, an independent set of breast adenocarcinomas was evaluated and demonstrated >2-fold upregulation of PARP1 in approximately 70% of primary breast adenocarcinomas, including TNBC, compared with syngeneic nonmalignant breast tissues. Immunohistochemistry (IHC) showed that upregulation of the PARP1 gene was consistent with increased protein expression in TNBC. These analyses suggest a potential biological role for PARP1 in several distinct malignancies, including TNBC. Further investigation of PARP1 as a biomarker for the therapeutic activity of PARP inhibitor-based therapy is warranted.

Dietary components and changes cause shifts in the gastrointestinal microbial ecology that can play a role in animal health and productivity. However, most information about the microbial populations in the gut of livestock species has not been quantitative. In the present study, we utilized a new molecular method, bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) that can perform diversity analyses of gastrointestinal bacterial populations. In the present study, cattle (n = 6) were fed a basal feedlot diet and were subsequently randomly assigned to 1 of 3 diets (n = 2 cows per diet). In each diet, 0, 25, or 50% of the concentrate portion of the ration was replaced with dried distillers grain (DDGS). Ruminal and fecal bacterial populations were different when animals were fed DDGS compared with controls; ruminal and fecal Firmicute:Bacteroidetes ratios were smaller (P = 0.07) in the 25 and 50% DDG diets compared with controls. Ruminal pH was decreased (P < 0.05) in ruminal fluid from cattle fed diets containing 50% compared with 0% DDGS. Using bTEFAP, the normal microbiota of cattle were examined using modern molecular methods to understand how diets affect gastrointestinal ecology and the gastrointestinal contribution of the microbiome to animal health and production.

A method was developed specifically to detect naturally occurring Listeria monocytogenes in meat because the traditional cold enrichment procedure was extremely slow and other procedures were ineffective. This method could identify beta-hemolytic Listeria colonies in 3-4 days. The use of a 2-stage enrichment, highly selective LPM agar, and a thin-layer horse blood agar plate for the detection of beta-hemolytic Listeria isolates are the important steps of this method. L. monocytogenes was recovered from 20 of 41 samples of frozen ground beef, 12 of 23 samples of pork sausage, and 7 of 22 samples of poultry. These results indicate that L. monocytogenes is common in raw meat and that this method is effective for its recovery.

Heavy metals can be enriched in living organisms and seriously endanger human health and the ecological environment, which has evolved into a significant global environmental problem. Based on summarizing the spatial distribution of heavy metals in the environment, this review introduces heavy metal detection technologies such as inductively coupled plasma mass spectrometry/atomic emission spectrometry, atomic absorption spectrometry, atomic fluorescence spectrometry, and laser-induced breakdown spectrometry. It summarizes their respective advantages, characteristics, and applicability. Besides, atmospheric pressure discharge plasma as a potential heavy metal detection technology is also introduced and discussed in this review. The current research mainly focuses on improving the analytical performance and optimizing the practical application. Furthermore, this review not only summarizes the advantages of atmospheric pressure discharge plasma in the field of element analysis but also summarizes the principal scientific and technical problems to be solved urgently.

BACKGROUND: Listeriosis can cause severe disease, especially in fetuses, neonates, older adults, and persons with certain immunocompromising and chronic conditions. We summarize US population-based surveillance data for invasive listeriosis from 2004 through 2009. METHODS: We analyzed Foodborne Diseases Active Surveillance Network (FoodNet) data for patients with Listeria monocytogenes isolated from normally sterile sites. We describe the epidemiology of listeriosis, estimate overall and specific incidence rates, and compare pregnancy-associated and nonpregnancy-associated listeriosis by age and ethnicity. RESULTS: A total of 762 listeriosis cases were identified during the 6-year reporting period, including 126 pregnancy-associated cases (17%), 234 nonpregnancy-associated cases(31%) in patients aged <65 years, and 400 nonpregnancy-associated cases (53%) in patients aged ≥ 65 years. Eighteen percent of all cases were fatal. Meningitis was diagnosed in 44% of neonates. For 2004-2009, the overall annual incidence of listeriosis varied from 0.25 to 0.32 cases per 100,000 population. Among Hispanic women, the crude incidence of pregnancy-associated listeriosis increased from 5.09 to 12.37 cases per 100,000 for the periods of 2004-2006 and 2007-2009, respectively; among non-Hispanic women, pregnancy-associated listeriosis increased from 1.74 to 2.80 cases per 100,000 for the same periods. Incidence rates of nonpregnancy-associated listeriosis in patients aged ≥ 65 years were 4-5 times greater than overall rates annually. CONCLUSIONS: Overall listeriosis incidence did not change significantly from 2004 through 2009. Further targeted prevention is needed, including food safety education and messaging (eg, avoiding Mexican-style cheese during pregnancy). Effective prevention among pregnant women, especially Hispanics, and older adults would substantially affect overall rates.

BACKGROUND: Listeriosis, a life-threatening foodborne illness caused by Listeria monocytogenes, affects approximately 2500 Americans annually. Between July and October 2002, an uncommon strain of L. monocytogenes caused an outbreak of listeriosis in 9 states. METHODS: We conducted case finding, a case-control study, and traceback and microbiological investigations to determine the extent and source of the outbreak and to propose control measures. Case patients were infected with the outbreak strain of L. monocytogenes between July and November 2002 in 9 states, and control patients were infected with different L. monocytogenes strains. Outcome measures included food exposure associated with outbreak strain infection and source of the implicated food. RESULTS: Fifty-four case patients were identified; 8 died, and 3 pregnant women had fetal deaths. The case-control study included 38 case patients and 53 control patients. Case patients consumed turkey deli meat much more frequently than did control patients (P = .008, by Wilcoxon rank-sum test). In the 4 weeks before illness, 55% of case patients had eaten deli turkey breast more than 1-2 times, compared with 28% of control patients (odds ratio, 4.5; 95% confidence interval, 1.3-17.1). Investigation of turkey deli meat eaten by case patients led to several turkey processing plants. The outbreak strain was found in the environment of 1 processing plant and in turkey products from a second. Together, the processing plants recalled > 30 million pounds of products. Following the outbreak, the US Department of Agriculture's Food Safety and Inspection Service issued new regulations outlining a L. monocytogenes control and testing program for ready-to-eat meat and poultry processing plants. CONCLUSIONS: Turkey deli meat was the source of a large multistate outbreak of listeriosis. Investigation of this outbreak helped guide policy changes designed to prevent future L. monocytogenes contamination of ready-to-eat meat and poultry products.

An epidemiological investigation was conducted to identify risk factors related to hygiene and husbandry practices which determine the introduction of Campylobacter spp. into broiler chicken flocks. All 176 broiler farms in an area in southeastern Norway participated in the study. Each farm was represented by one flock selected at random during a one-year period. The flocks were examined for campylobacter colonization at slaughter, and the flock managers were subsequently interviewed about hygiene and husbandry practices. Campylobacter spp. were recovered from 32 (18%) of the flocks. The proportion of colonized flocks varied geographically and seasonally with a peak in the autumn. The following variables were found to be independently associated with an increased risk of campylobacter colonization using logistic regression analysis: (i) feeding the broilers undisinfected water (odds ratio (OR) = 3.42, P = 0.045), (ii) tending other poultry prior to entering the broiler house (OR = 6.43, P = 0.007), (iii) tending pigs before entering the house (OR = 4.86, P = 0.037), (iv) geographic region (Hedmark versus Ostfold county) (OR = 2.91, P = 0.023, (v) season (autumn versus other seasons) (OR = 3.43; P = 0.008). Presence of rats on the farm was associated with an increased risk, but this factor did not reach statistical significance (OR = 3.96, P = 0.083). Preventive measures should include disinfection of drinking water and strict hygienic routines when the farm workers enter the rearing room. The results indicate that disinfection of drinking water is the preventive measure most likely to have the greatest impact on the prevalence of campylobacter among broiler chicken flocks in the study area (population attributable fraction = 0.53).

We used molecular subtyping to investigate an outbreak of listeriosis involving residents of 24 US states. We defined a case as infection with Listeria monocytogenes serotype 4b yielding one of several closely related patterns when subtyped by pulsed-field gel electrophoresis. Patients infected with strains yielding different patterns were used as controls. A total of 108 cases were identified with 14 associated deaths and four miscarriages or stillbirths. A case-control study implicated meat frankfurters as the likely source of infection (OR 17.3, 95% CI 2.4-160). The outbreak ended abruptly following a manufacturer-issued recall, and the outbreak strain was later detected in low levels in the recalled product. A second strain was recovered at higher levels but was not associated with human illness. Our findings suggest that L. monocytogenes strains vary widely in virulence and confirm that large outbreaks can occur even when only low levels of contamination are detected in sampled food. Standardized molecular subtyping and coordinated, multi-jurisdiction investigations can greatly facilitate detection and control of listeriosis outbreaks.

The crop is a known source of Salmonella and Campylobacter contamination. We evaluated the use of selected organic acids (0.5% acetic, lactic, or formic) in drinking water during a simulated 8-h pretransport feed withdrawal (FW). Salmonella typhimurium was recovered from 53/100 control crops and from 45/100 of crops from acetic acid-treated broilers. However, treatment with lactic acid (31/100) or formic acid (28/76) caused significant (P < 0.05) reduction in incidence. Reductions of recovered incidence were also associated with reduced numbers of S. typhimurium recovered (e.g., control, log 1.45 cfu/crop; lactic acid, 0.79 cfu/crop). In an additional commercial farm study, broilers were provided 0.44% lactic acid during a 10-h FW (4 h on the farm and 6 h transport) and pre-FW crop, post-FW crop, and pre-chill carcass wash samples were collected for Campylobacter and Salmonella detection. Crop contamination with Salmonella was significantly reduced by lactic acid treatment (6/175) as compared with controls (29/175). Importantly, Salmonella isolation incidence in prechill carcass rinses was significantly reduced by 52.4% with the use of lactic acid (26/175 vs. 55/176). Crop contamination with Campylobacter was significantly reduced by lactic acid treatment (62.3%) as compared with the controls (85.1%). Lactic acid also reduced the incidence of Campylobacter found on pre-chill carcass rinses by 14.7% compared with the controls. These studies suggest that incorporation of lactic acid in the drinking water during pretransport FW may reduce Salmonella and Campylobacter contamination of crops and broiler carcasses at processing.

; if clinical laboratories do not, the burden of culturing falls to state public health laboratories, which might not be able to absorb that burden as the adoption of these tests increases (2). Strategies are needed to preserve access to bacterial isolates for further characterization and to determine the effect of changing trends in testing practices on surveillance.

Drug-resistant bacterial infections pose a serious and growing public health threat globally. In this review, we describe the role of the National Antimicrobial Resistance Monitoring System (NARMS) in providing data that help address the resistance problem and show how such a program can have broad positive impacts on public health. NARMS was formed two decades ago to help assess the consequences to human health arising from the use of antimicrobial drugs in food animal production in the United States. A collaboration among the Centers for Disease Control and Prevention, the U.S. Food and Drug Administration, the United States Department of Agriculture, and state and local health departments, NARMS uses an integrated "One Health" approach to monitor antimicrobial resistance in enteric bacteria from humans, retail meat, and food animals. NARMS has adapted to changing needs and threats by expanding surveillance catchment areas, examining new isolate sources, adding bacteria, adjusting sampling schemes, and modifying antimicrobial agents tested. NARMS data are not only essential for ensuring that antimicrobial drugs approved for food animals are used in ways that are safe for human health but they also help address broader food safety priorities. NARMS surveillance, applied research studies, and outbreak isolate testing provide data on the emergence of drug-resistant enteric bacteria; genetic mechanisms underlying resistance; movement of bacterial populations among humans, food, and food animals; and sources and outcomes of resistant and susceptible infections. These data can be used to guide and evaluate the impact of science-based policies, regulatory actions, antimicrobial stewardship initiatives, and other public health efforts aimed at preserving drug effectiveness, improving patient outcomes, and preventing infections. Many improvements have been made to NARMS over time and the program will continue to adapt to address emerging resistance threats, changes in clinical diagnostic practices, and new technologies, such as whole genome sequencing.

Microgreens have gained increasing popularity as food ingredients in recent years because of their high nutritional value and diverse sensorial characteristics. Microgreens are edible seedlings including vegetables and herbs, which have been used, primarily in the restaurant industry, to embellish cuisine since 1996. The rapidly growing microgreen industry faces many challenges. Microgreens share many characteristics with sprouts, and while they have not been associated with any foodborne illness outbreaks, they have recently been the subject of seven recalls. Thus, the potential to carry foodborne pathogens is there, and steps can and should be taken during production to reduce the likelihood of such incidents. One major limitation to the growth of the microgreen industry is the rapid quality deterioration that occurs soon after harvest, which keeps prices high and restricts commerce to local sales. Once harvested, microgreens easily dehydrate, wilt, decay and rapidly lose certain nutrients. Research has explored preharvest and postharvest interventions, such as calcium treatments, modified atmopsphere packaging, temperature control, and light, to maintain quality, augment nutritional value, and extend shelf life. However, more work is needed to optimize both production and storage conditions to improve the safety, quality, and shelf life of microgreens, thereby expanding potential markets.

Whole-genome sequencing (WGS) is increasingly used by food regulatory and public health agencies in the United States to facilitate the detection, investigation, and control of foodborne bacterial outbreaks, and food regulatory and other activities in support of food safety. WGS has added a level of precision to the surveillance leading to faster and more efficient decision making in the preparedness and response to foodborne infections. In this review, we report the history of WGS technology at the Centers for Disease Control & Prevention (CDC), the Food and Drug Administration (FDA), and the United States Department of Agriculture's Food Safety and Inspection Service (USDA/FSIS) as it applies to food safety. The basic principle of the method, the analysis, and interpretation of the data are explained as is its major strengths and limitations. We also describe the benefits and possibilities of the WGS technology to the food industry throughout the farm-to-fork continuum and the prospects of metagenomic sequencing applied directly to the sample specimen with or without pre-enrichment culture.

Listeria monocytogenes is a serious food-borne pathogen that can cause invasive disease in humans and other animals and has been the leading cause of food recalls due to microbiological concerns in recent years. In order to test hypotheses regarding L. monocytogenes lineage composition, evolution, ecology, and taxonomy, a robust intraspecific phylogeny was developed based on prfA virulence gene cluster sequences from 113 L. monocytogenes isolates. The results of the multigene phylogenetic analyses confirm that L. monocytogenes comprises at least three evolutionary lineages, demonstrate that lineages most frequently (lineage 1) and least frequently (lineage 3) associated with human listeriosis are sister-groups, and reveal for the first time that the human epidemic associated serotype 4b is prevalent among strains from lineage 1 and lineage 3. In addition, a PCR-based test for lineage identification was developed and used in a survey of food products demonstrating that the low frequency of association between lineage 3 isolates and human listeriosis cases likely reflects rarity of exposure and not reduced virulence for humans as has been previously suggested. However, prevalence data do suggest lineage 3 isolates may be better adapted to the animal production environment than the food-processing environment. Finally, analyses of haplotype diversity indicate that lineage 1 has experienced a purge of genetic variation that was not observed in the other lineages, suggesting that the three L. monocytogenes lineages may represent distinct species within the framework of the cohesion species concept.

In investigations on three outbreaks of Bacillus cereus food poisoning in Spain and The Netherlands, the causative strains grew within a temperature range of 4-37 degrees C, but not at 43 degrees C. Such psychrotrophic types were found to occur in various dairy products (including ca 25% of 35 samples of pasteurized milk) and some mousses and cook/chill meals. Growth of and enterotoxin production by psychrotrophic B. cereus could be prevented by temperatures below 4 degrees C and pH-values not exceeding 5.0.