CRISPR/Cas9 targeted mutations of OsDSG1 gene enhanced salt tolerance in rice.

Salinization is one of the leading causes of arable land shrinkage and rice yield decline, recently. Therefore, developing and utilizing salt-tolerant rice varieties have been seen as a crucial and urgent strategy to reduce the effects of saline intrusion and protect food security worldwide. In the current study, the CRISPR/Cas9 system was utilized to induce targeted mutations in the coding sequence of the OsDSG1, a gene involved in the ubiquitination pathway and the regulation of biochemical reactions in rice. The CRISPR/Cas9-induced mutations of the OsDSG1 were generated in a local rice cultivar and the mutant inheritance was validated at different generations. The OsDSG1 mutant lines showed an enhancement in salt tolerance compared to wild type plants at both germination and seedling stages indicated by increases in plant height, root length, and total fresh weight as well as the total chlorophyll and relative water contents under the salt stress condition. In addition, lower proline and MDA contents were observed in mutant rice as compared to wild type plants in the presence of salt stress. Importantly, no effect on seed germination and plant growth parameters was recorded in the CRISRP/Cas9-induced mutant rice under the normal condition. This study again indicates the involvement of the OsDSG1 gene in the salt resistant mechanism in rice and provides a potential strategy to enhance the tolerance of local rice varieties to the salt stress.

Evaluating a Low-Cost Disaster Preparedness Simulation for Prelicensure Nursing Students.

OBJECTIVE: This innovation in simulation evaluated the effectiveness of a time sensible, low-cost simulation on prelicensure nursing students' knowledge and confidence in responding to public health emergencies. METHOD: One hundred eighty-two nursing students, in groups of 5, participated in a 75-min emergency preparedness disaster simulation. A mixed methods design was used to evaluate students' knowledge and confidence in disaster preparedness, and satisfaction with the simulation. RESULTS: Students reported an increase in knowledge and confidence following the disaster simulation and satisfaction with the experience. CONCLUSIONS: Prelicensure nursing programs can replicate this low cost, time sensible disaster simulation to effectively educate students in emergency preparedness.

Disrupting Sc-uORFs of a transcription factor bZIP1 using CRISPR/Cas9 enhances sugar and amino acid contents in tomato (Solanum lycopersicum).

MAIN CONCLUSION: Induced mutations in the SC-uORF of the tomato transcription factor gene SlbZIP1 by the CRISPR/Cas9 system led to the high accumulation of sugar and amino acid contents in tomato fruits. Tomato (Solanum lycopersicum) is one of the most popular and consumed vegetable crops in the world. Among important traits for tomato improvement such as yield, biotic and abiotic resistances, appearance, post-harvest shelf life and fruit quality, the last one seems to face more challenges because of its genetic and biochemical complexities. In this study, a dual-gRNAs CRISPR/Cas9 system was developed to induce targeted mutations in uORF regions of the SlbZIP1, a gene involved in the sucrose-induced repression of translation (SIRT) mechanism. Different induced mutations in the SlbZIP1-uORF region were identified at the T0 generation, stably transferred to the offspring, and no mutation was found at potential off-target sites. The induced mutations in the SlbZIP1-uORF region affected the transcription of SlbZIP1 and related genes in sugar and amino acid biosynthesis. Fruit component analysis showed significant increases in soluble solid, sugar and total amino acid contents in all SlbZIP1-uORF mutant lines. The accumulation of sour-tasting amino acids, including aspartic and glutamic acids, raised from 77 to 144%, while the accumulation of sweet-tasting amino acids such as alanine, glycine, proline, serine, and threonine increased from 14 to 107% in the mutant plants. Importantly, the potential SlbZIP1-uORF mutant lines with desirable fruit traits and no impaired effect on plant phenotype, growth and development were identified under the growth chamber condition. Our result indicates the potential utility of the CRISPR/Cas9 system for fruit quality improvement in tomato and other important crops.

Impact of antimicrobial stewardship intervention in clean and clean-contaminated surgical procedures at a Vietnamese national hospital.

OBJECTIVES: We investigated the characteristics of prophylactic antimicrobial use in clean and clean-contaminated surgical procedures and assessed the efficacy of a prophylactic antimicrobial stewardship intervention at Thong Nhat Hospital, Ho Chi Minh City, Vietnam. METHODS: A cross-sectional study was conducted on 354 patients who underwent either clean or clean-contaminated surgical procedures at Thong Nhat Hospital. Eligible patients were classified with respect to three periods of intervention from 2017 to 2020. Data collection included surgical procedures, patient characteristics, and prophylactic antimicrobial usage. We determined the efficacy of antimicrobial stewardship intervention based on comparisons among the primary outcome (the appropriateness of prophylactic antimicrobials) and secondary outcomes (postoperative antimicrobial prophylaxis (AP) prolongation, length of postoperative hospital stay, and cost of antimicrobials). RESULTS: The mean age of patients in periods 1, 2, and 3 was 54.5 ± 16.6, 50.2 ± 16.5, and 52.8 ± 17.3 years, respectively, with an overall male/female ratio of 1.1/1. No significant differences were detected in basic patient characteristics during the three periods. Majority of the surgical procedures were clean (56%-59%) and scheduled (85%-86%). Prophylactic antimicrobial stewardship intervention enhanced AP appropriateness (by 12.7%, 12.7%, and 39.0% in periods 1, 2, and 3, respectively, p < 0.001), decreased postoperative prophylactic antimicrobial duration [3.0 (0-6), 1.5 (0-5), and 0.0 (0-1) days, respectively, p < 0.001], and reduced average antimicrobial expenses (p < 0.001). CONCLUSIONS: The prophylactic antimicrobial stewardship interventions introduced at Thong Nhat Hospital had several positive impacts on the appropriateness of prophylactic antimicrobial use and treatment costs.

Agritourism and Kidding Season: A Large Outbreak of Human Shiga Toxin-Producing Escherichia coli O157 (STEC O157) Infections Linked to a Goat Dairy Farm-Connecticut, 2016.

The objective of this study was to determine sources of Shiga toxin-producing Escherichia coli O157 (STEC O157) infection among visitors to Farm X and develop public health recommendations. A case-control study was conducted. Case-patients were defined as the first ill child (aged <18 years) in the household with laboratory-confirmed STEC O157, or physician-diagnosed hemolytic uremic syndrome with laboratory confirmation by serology, who visited Farm X in the 10 days prior to illness. Controls were selected from Farm X visitors aged <18 years, without symptoms during the same time period as case-patients. Environment and animal fecal samples collected from Farm X were cultured; isolates from Farm X were compared with patient isolates using whole genome sequencing (WGS). Case-patients were more likely than controls to have sat on hay bales at the doe barn (adjusted odds ratio: 4.55; 95% confidence interval: 1.41-16.13). No handwashing stations were available; limited hand sanitizer was provided. Overall, 37% (29 of 78) of animal and environmental samples collected were positive for STEC; of these, 62% (18 of 29) yielded STEC O157 highly related by WGS to patient isolates. STEC O157 environmental contamination and fecal shedding by goats at Farm X was extensive. Farms should provide handwashing stations with soap, running water, and disposable towels. Access to animal areas, including animal pens and enclosures, should be limited for young children who are at risk for severe outcomes from STEC O157 infection. National recommendations should be adopted to reduce disease transmission.

Genetic Diversity of Non-O157 Shiga Toxin-Producing Escherichia coli Recovered From Patients in Michigan and Connecticut.

Shiga toxin-producing Escherichia coli (STEC) are important foodborne pathogens and non-O157 serotypes have been gradually increasing in frequency. The non-O157 STEC population is diverse and is often characterized using serotyping and/or multilocus sequence typing (MLST). Although spacers within clustered regularly interspaced repeat (CRISPR) regions were shown to comprise horizontally acquired DNA elements, this region does not actively acquire spacers in STEC. Hence, it is useful for further characterizing non-O157 STEC and examining relationships between strains. Our study goal was to evaluate the genetic relatedness of 41 clinical non-O157 isolates identified in Michigan between 2001 and 2005 while comparing to 114 isolates from Connecticut during an overlapping time period. Whole genome sequencing (WGS) was performed, and sequences were extracted for serotyping, MLST and CRISPR analysis. Phylogenetic analysis of MLST and CRISPR data was performed using the Neighbor joining and unweighted pair group method with arithmetic mean (UPGMA) algorithms, respectively. In all, 29 serogroups were identified; eight were unique to Michigan and 13 to Connecticut. "Big-six" serogroup frequencies were similar by state (Michigan: 73.2%, Connecticut: 81.6%), though STEC O121 was not found in Michigan. The distribution of sequence types (STs) and CRISPR profiles was also similar across states. Interestingly, big-six serogroups such as O103 and O26, grouped into different STs located on distinct branches of the phylogeny, further confirming that serotyping alone is not adequate for evaluating strain relatedness. Comparatively, the CRISPR analysis identified 361 unique spacers that grouped into 80 different CRISPR profiles. CRISPR spacers 231 and 317 were isolated from 79.2% (n = 118) and 59.1% (n = 88) of strains, respectively, regardless of serogroup and ST. Spacer profiles clustered according to the MLST analysis, though some discrepancies were noted. Indeed, use of both MLST and CRISPR typing enhanced the discriminatory power when compared to the use of each tool separately. These data highlight the genetic diversity of clinical STEC from different locations and show that CRISPR profiling can be used alongside MLST to discriminate related strains. Use of targeted sequencing approaches are particularly helpful for sites without WGS capabilities and can help define which strains require additional characterization using more discriminatory methods.

Characteristics of Clusters of Salmonella and Escherichia coli O157 Detected by Pulsed-Field Gel Electrophoresis that Predict Identification of Outbreaks.

INTRODUCTION: Molecular subtyping of pathogens is critical for foodborne disease outbreak detection and investigation. Many clusters initially identified by pulsed-field gel electrophoresis (PFGE) are not confirmed as point-source outbreaks. We evaluated characteristics of clusters that can help prioritize investigations to maximize effective use of limited resources. MATERIALS AND METHODS: A multiagency collaboration (FoodNet) collected data on Salmonella and Escherichia coli O157 clusters for 3 years. Cluster size, timing, extent, and nature of epidemiologic investigations were analyzed to determine associations with whether the cluster was identified as a confirmed outbreak. RESULTS: During the 3-year study period, 948 PFGE clusters were identified; 849 (90%) were Salmonella and 99 (10%) were E. coli O157. Of those, 192 (20%) were ultimately identified as outbreaks (154 [18%] of Salmonella and 38 [38%] of E. coli O157 clusters). Successful investigation was significantly associated with larger cluster size, more rapid submission of isolates (e.g., for Salmonella, 6 days for outbreaks vs. 8 days for nonoutbreaks) and PFGE result reporting to investigators (16 days vs. 29 days, respectively), and performance of analytic studies (completed in 33% of Salmonella outbreaks vs. 1% of nonoutbreaks) and environmental investigations (40% and 1%, respectively). Intervals between first and second cases in a cluster did not differ significantly between outbreaks and nonoutbreaks. CONCLUSIONS: Molecular subtyping of pathogens is a rapidly advancing technology, and successfully identifying outbreaks will vary by pathogen and methods used. Understanding criteria for successfully investigating outbreaks is critical for efficiently using limited resources.

Outbreak of Salmonella enterica serotype Infantis infection in humans linked to dry dog food in the United States and Canada, 2012.

CASE DESCRIPTION--In April 2012, Salmonella enterica serotype Infantis was detected in an unopened bag of dry dog food collected during routine retail surveillance. PulseNet, a national bacterial subtyping network, identified humans with Salmonella Infantis infection with the same genetic fingerprint as the dog food sample. CLINICAL FINDINGS--An outbreak investigation identified 53 ill humans infected with the outbreak strain during January 1 to July 5, 2012, in 21 states and 2 provinces in Canada; 20 (38%) were children ≤ 2 years old, and 12 of 37 (32%) were hospitalized. Of 21 ill people who remembered the dog food brand, 12 (57%) reported a brand produced at a plant in Gaston, SC. Traceback investigations also identified that plant. The outbreak strain was isolated from bags of dry dog food and fecal specimens obtained from dogs that lived with ill people and that ate the implicated dry dog food. TREATMENT AND OUTCOME--The plant was closed temporarily for cleaning and disinfection. Sixteen brands involving > 27,000 metric tons (> 30,000 tons) of dry dog and cat food were recalled. Thirty-one ill dogs linked to recalled products were reported through the FDA consumer complaint system. CLINICAL RELEVANCE-- A one-health collaborative effort on epidemiological, laboratory, and traceback investigations linked dry dog foods produced at a plant to illnesses in dogs and humans. More efforts are needed to increase awareness among pet owners, health-care professionals, and the pet food industry on the risk of illness in pets and their owners associated with dry pet foods and treats.

Salmonella enterica serotype enteritidis outbreak at a long-term care facility, Connecticut, 2012.

In May of 2012, the Connecticut Department of Public Health (DPH) was notified of three hospitalized residents of a long-term care facility (LTCF) who had gastrointestinal illness, one of whom had a stool culture positive for Salmonella enterica. A multiagency outbreak investigation was initiated and identified a total of 21 possible salmonellosis cases; nine were culture-confirmed Salmonella serotype Enteritidis with an indistinguishable pulsed-field gel electrophoresis pattern (PFGE). This report describes the epidemiologic, environmental, and laboratory investigation conducted as part of DPH's response. Undercooked raw shell eggs were the likely source of infection. This investigation reemphasizes the vulnerabilityof certain populations to severe illness from Salmonella and further stresses previous recommendations in the literature to use only pasteurized egg products in long-term care and other health care facilities.

Contributing factors in restaurant-associated foodborne disease outbreaks, FoodNet sites, 2006 and 2007.

An estimated 48 million cases of foodborne illness occur each year in the United States, resulting in approximately 128,000 hospitalizations and 3,000 deaths. Over half of all foodborne disease outbreaks reported to the Centers for Disease Control and Prevention are associated with eating in restaurants or delicatessens. We reviewed data from restaurant-associated foodborne disease outbreaks to better understand the factors that contribute to these outbreaks. Data on restaurant-associated foodborne disease outbreaks reported by sites participating in the Foodborne Diseases Active Surveillance Network (FoodNet) were analyzed to characterize contributing factors reported in foodborne disease outbreaks and the levels of evidence used to identify these factors. Of 457 foodborne disease outbreaks reported in 2006 and 2007 by FoodNet sites, 300 (66%) were restaurant associated, and of these 295 (98%) had at least one reported contributing factor. One to nine (with a median of two) contributing factors were reported per outbreak. Of the 257 outbreaks with a single etiology reported, contributing factors associated with food worker health and hygiene were reported for 165 outbreaks (64%), factors associated with food preparation practices within the establishment were reported for 88 outbreaks (34%), and factors associated with contamination introduced before reaching the restaurant were reported for 56 outbreaks (22%). The pronounced role of food workers in propagating outbreaks makes it clear that more work is needed to address prevention at the local level. Food workers should be instructed not to prepare food while ill to prevent the risk of transmitting pathogens.

Characteristics of foodborne disease outbreak investigations conducted by Foodborne Diseases Active Surveillance Network (FoodNet) sites, 2003-2008.

BACKGROUND: A mean of ≥ 1000 foodborne disease outbreaks (FBDOs) causing ≥ 20,000 illnesses are reported to the Centers for Disease Control and Prevention (CDC) annually. We evaluated characteristics of successful outbreak investigations (ie, those that identified an etiologic agent or food vehicle) in the Foodborne Diseases Active Surveillance Network (FoodNet). METHODS: FBDOs were defined as the occurrence of ≥ 2 cases of a similar illness resulting from ingestion of a common food. FBDOs reported to CDC Foodborne Disease Outbreak Surveillance System during 2003-2008 with FoodNet supplemental data available were included in the analyses. RESULTS: Data regarding 1200 FBDOs were available. An etiologic agent was confirmed in 715 (60%); a food vehicle was identified in 387 (32%). At least 4 fecal specimens were collected in 425 of 639 outbreaks (67%) with a confirmed etiologic agent and 48 of 232 (21%) without a confirmed etiologic agent (odds ratio [OR], 7.6; 95% confidence interval [CI], 5.3-10.9). A food vehicle was identified in 314 (47%) of 671 outbreaks investigated using a case-control or cohort study, compared with only 73 (14%) of 529 outbreaks investigated by using other methods (OR, 5.5; 95% CI, 4.1-7.3). At least 1 barrier affecting the success of the investigation was reported for 655 outbreaks, including too few patients (n = 172; 26%), too few stool specimens (n = 167; 25%), and too few control subjects (n = 152; 23%). CONCLUSIONS: Etiologic agent and vehicle are frequently undetermined in FBDOs. Greater emphasis on fecal specimen collection and overcoming barriers to pursuing analytic epidemiologic studies can improve ascertainment of these factors.

Asymmetric type F botulism with cranial nerve demyelination.

We report a case of type F botulism in a patient with bilateral but asymmetric neurologic deficits. Cranial nerve demyelination was found during autopsy. Bilateral, asymmetric clinical signs, although rare, do not rule out botulism. Demyelination of cranial nerves might be underrecognized during autopsy of botulism patients.

Salmonella typhimurium infections associated with peanut products.

BACKGROUND: Contaminated food ingredients can affect multiple products, each distributed through various channels and consumed in multiple settings. Beginning in November 2008, we investigated a nationwide outbreak of salmonella infections. METHODS: A case was defined as laboratory-confirmed infection with the outbreak strain of Salmonella Typhimurium occurring between September 1, 2008, and April 20, 2009. We conducted two case-control studies, product "trace-back," and environmental investigations. RESULTS: Among 714 case patients identified in 46 states, 166 (23%) were hospitalized and 9 (1%) died. In study 1, illness was associated with eating any peanut butter (matched odds ratio, 2.5; 95% confidence interval [CI], 1.3 to 5.3), peanut butter-containing products (matched odds ratio, 2.2; 95% CI, 1.1 to 4.7), and frozen chicken products (matched odds ratio, 4.6; 95% CI, 1.7 to 14.7). Investigations of focal clusters and single cases associated with nine institutions identified a single institutional brand of peanut butter (here called brand X) distributed to all facilities. In study 2, illness was associated with eating peanut butter outside the home (matched odds ratio, 3.9; 95% CI, 1.6 to 10.0) and two brands of peanut butter crackers (brand A: matched odds ratio, 17.2; 95% CI, 6.9 to 51.5; brand B: matched odds ratio, 3.6; 95% CI, 1.3 to 9.8). Both cracker brands were made from brand X peanut paste. The outbreak strain was isolated from brand X peanut butter, brand A crackers, and 15 other products. A total of 3918 peanut butter-containing products were recalled between January 10 and April 29, 2009. CONCLUSIONS: Contaminated peanut butter and peanut products caused a nationwide salmonellosis outbreak. Ingredient-driven outbreaks are challenging to detect and may lead to widespread contamination of numerous food products.

Ten-year trends and risk factors for non-O157 Shiga toxin-producing Escherichia coli found through Shiga toxin testing, Connecticut, 2000-2009.

BACKGROUND: The epidemiology over time of non-O157 Shiga toxin-producing Escherichia coli (STEC) is unknown. Since 1999, increasing numbers of laboratories in Connecticut have been testing for ST rather than culturing for O157, enabling identification of non-O157 STEC. METHODS: Beginning in 2000, Connecticut laboratories were required to submit ST-positive broths to the State Laboratory for isolation and typing of STEC. The ratio of non-O157:O157 from laboratories conducting ST testing was used to determine state-level estimates for non-O157 STEC. Patients with STEC were interviewed for exposure factors in the 7 days preceding illness. Incidence trends, clinical features, and epidemiology of non-O157 and O157 STEC infections were compared. RESULTS: From 1 January 2000 through 31 December 2009, ST testing detected 392 (59%) of 663 reported STEC infections; 229 (58%) of the isolates were non-O157. The estimated incidence of STEC infection decreased by 34%. O157 and the top 4 non-O157 serogroups, O111, O103, O26, and O45, were a stable percentage of all STEC isolates over the 10-year period. Bloody diarrhea, hospitalization, and hemolytic uremic syndrome were more common in patients with O157 STEC than in patients with non-O157 STEC infection. Exposure risks of patients with non-O157 STEC infection differed from those of patients with O157 STEC infection primarily in international travel (15.3% vs 2.5%; P < .01). Non-O157 types differed from each other with respect to several epidemiologic and exposure features. CONCLUSIONS: Both O157 and non-O157 STEC infection incidence decreased from 2000 through 2009. Although infection due to O157 is the most common and clinically severe STEC infection, it accounts for a minority of all clinically significant STEC infections. STEC appear to be a diverse group of organisms that have some differences as well as many epidemiologic and exposure features in common.

The evolving epidemiology of hepatitis a in the United States: incidence and molecular epidemiology from population-based surveillance, 2005-2007.

BACKGROUND: The incidence of hepatitis A virus (HAV) disease is the lowest ever in the United States. We describe recent incidence and characteristics of cases of HAV disease from 6 US sites conducting hepatitis surveillance in the Emerging Infections Program. METHODS: Health departments conducted enhanced, population-based surveillance for HAV from 2005 through 2007. Demographic and risk factor data were collected on suspected cases (persons with a positive IgM anti-HAV result) using a standard form. Remnant serum specimens from a convenience sample of cases were tested by polymerase chain reaction, followed by sequencing the 315-nucleotide segment of the VP1-P2B junction. RESULTS: There were 1156 HAV cases reported during 2005 through 2007. The combined population under surveillance was 29.8 million in 2007. The overall annual incidence rate was 1.3 per 100 000 population (range by site, 0.7-2.3). Of reported cases, 53.4% were male, 42.4% were white, 44.7% were aged 15 to 39 years, and 91.4% resided in urban areas. Reported risk factors were international travel (45.8%), contact with a case (14.8%), employee or child in a daycare center (7.6%), exposure during a food or waterborne common-source outbreak (7.2%), illicit drug use (4.3%), and men who had sex with men (3.9%). Genotypes among the 271 case specimens were IA (87.8%), IB (11.4%), and IIIA (0.7%). Of the 271 polymerase chain reaction-positive specimens, 131 (48.3%) were from cases reporting travel or exposure to a traveler; 58 of the 131 cases reported travel to Mexico, and 53 of the 58 were within the US-IA(1) cluster. CONCLUSIONS: International travel was the predominant risk factor for HAV transmission. Health care providers should encourage vaccination of at-risk travelers.

Outbreak of Escherichia coli O157 associated with raw milk, Connecticut, 2008.

BACKGROUND: In Connecticut, despite hazards of raw milk consumption, attempts to ban raw milk sales have been unsuccessful. In July 2008, 2 children experienced Escherichia coli O157-associated hemolytic uremic syndrome (HUS) after consuming raw milk purchased at a retail market and a farm (farm X). We investigated to determine the outbreak source and control measures. METHODS: Confirmed cases were HUS diagnosis or E. coli O157:NM infections with isolates matching outbreak strains among patients during June to July 2008. Probable cases were diarrheal illness among farm X customers during the same period. We conducted case-control studies to determine the source of E. coli O157 exposure and assess for dose-response relation between illness and frequency of raw milk consumption. Farm X dairy practices were evaluated; stool specimens of humans and animals were cultured for E. coli O157. Staff time and laboratory and medical costs were calculated. RESULTS: We identified 14 cases (7 confirmed). Five (36%) case patients required hospitalization; 3 (21%) experienced HUS. No deaths were reported. Raw milk consumption was associated with illness (P = .008); a dose-response relation was demonstrated (P = .01). Dairy practices reflected industry standards. E. coli O157:NM outbreak strains were isolated from stool specimens of 6 case patients and 1 milking cow. The total estimated outbreak cost was $413,402. CONCLUSIONS: Farm X's raw milk was the outbreak source despite no violations of current raw milk regulatory standards. This outbreak resulted in substantial costs and proposed legislation to prohibit nonfarm retail sale, strengthen advisory labels, and increase raw milk testing for pathogens.

Chicken salad as the source of a case of Listeria monocytogenes infection in Connecticut.

Listeriosis is a severe infection with high morbidity and mortality. We report a fatal case of listeriosis in a patient with a history of Crohn's disease who consumed chicken salad purchased from a retail food establishment before developing listeriosis. As part of the regulatory testing programs, the U.S. Department of Agriculture Food Safety and Inspection Service and the Florida Department of Agriculture and Consumer Affairs found that chicken products from a single food-production establishment were contaminated with Listeria monocytogenes, resulting in a product recall. The case patient's Listeria isolate was subtyped by pulsed-field gel electrophoresis (PFGE) and matched the Listeria isolates from the recalled chicken products. Identification of the source of Listeria involved collaboration among two state public health laboratories and epidemiologists and state and federal regulatory agencies. PFGE typing can be used to reveal correlations between clusters of human illness and contaminated food products and to rapidly identify sources of Listeria infection to allow implementation of corrective actions at both the state and national levels.