Use of whole genome sequencing to complement characterisation of a typhoid fever outbreak among a Marshallese community: Oklahoma, 2015.

Typhoid fever is an illness caused by Salmonella enterica serotype Typhi. In developing regions, it affects an estimated 20 million people annually, causing 200 000 deaths. Although uncommon, cases occur in the USA each year, predominantly due to international travel. During February 2015, the Oklahoma State Department of Health (OSDH) detected an outbreak of typhoid fever among residents of northwestern Oklahoma. OSDH conducted case-patient interviews to identify the source and symptomatic contacts. Whole genome sequencing (WGS) was performed to characterise the genetic relatedness of isolates among the four outbreak-associated pulsed-field gel electrophoresis (PFGE) patterns. We identified 38 cases, 25 confirmed and 13 probable, in two states. WGS revealed a 0-10 single-nucleotide polymorphism variation between isolates. Although we were unable to determine the source, almost all case-patients were members of the Marshallese community that attended a common event in Oklahoma, or were contacts to a confirmed case. This is the largest outbreak of typhoid fever in the USA since 1989, and first to apply WGS to complement interpretation of PFGE results during a typhoid fever outbreak investigation. This investigation illustrates the potential risk of outbreaks among communities comprised of international populations from regions where typhoid fever remains endemic.

Flea market finds and global exports: Four multistate outbreaks of human Salmonella infections linked to small turtles, United States-2015.

Zoonotic transmission of Salmonella infections causes an estimated 11% of salmonellosis annually in the United States. This report describes the epidemiologic, traceback and laboratory investigations conducted in the United States as part of four multistate outbreaks of Salmonella infections linked to small turtles. Salmonella isolates indistinguishable from the outbreak strains were isolated from a total of 143 ill people in the United States, pet turtles, and pond water samples collected from turtle farm A, as well as ill people from Chile and Luxembourg. Almost half (45%) of infections occurred in children aged <5 years, underscoring the importance of the Centers for Disease Control and Prevention recommendation to keep pet turtles and other reptiles out of homes and childcare settings with young children. Although only 43% of the ill people who reported turtle exposure provided purchase information, most small turtles were purchased from flea markets or street vendors, which made it difficult to locate the vendor, trace the turtles to a farm of origin, provide education and enforce the United States federal ban on the sale and distribution of small turtles. These outbreaks highlight the importance of improving public awareness and education about the risk of Salmonella from small turtles not only in the United States but also worldwide.

Next-generation sequencing technologies and their application to the study and control of bacterial infections.

BACKGROUND: With the efficiency and the decreasing cost of next-generation sequencing, the technology is being rapidly introduced into clinical and public health laboratory practice. AIMS: The historical background and principles of first-, second- and third-generation sequencing are described, as are the characteristics of the most commonly used sequencing instruments. SOURCES: Peer-reviewed literature, white papers and meeting reports. CONTENT AND IMPLICATIONS: Next-generation sequencing is a technology that could potentially replace many traditional microbiological workflows, providing clinicians and public health specialists with more actionable information than hitherto achievable. Examples of the clinical and public health uses of the technology are provided. The challenge of comparability of different sequencing platforms is discussed. Finally, the future directions of the technology integrating it with laboratory management and public health surveillance systems, and moving it towards performing sequencing directly from the clinical specimen (metagenomics), could lead to yet another fundamental transformation of clinical diagnostics and public health surveillance.

Multistate outbreak of listeriosis caused by imported cheese and evidence of cross-contamination of other cheeses, USA, 2012.

Listeria monocytogenes is a foodborne pathogen that can cause bacteraemia, meningitis, and complications during pregnancy. In July 2012, molecular subtyping identified indistinguishable L. monocytogenes isolates from six patients and two samples of different cut and repackaged cheeses. A multistate outbreak investigation was initiated. Initial analyses identified an association between eating soft cheese and outbreak-related illness (odds ratio 17·3, 95% confidence interval 2·0-825·7) but no common brand. Cheese inventory data from locations where patients bought cheese and an additional location where repackaged cheese yielded the outbreak strain were compared to identify cheeses for microbiological sampling. Intact packages of imported ricotta salata yielded the outbreak strain. Fourteen jurisdictions reported 22 cases from March-October 2012, including four deaths and a fetal loss. Six patients ultimately reported eating ricotta salata; another reported eating cheese likely cut with equipment also used for contaminated ricotta salata, and nine more reported eating other cheeses that might also have been cross-contaminated. An FDA import alert and US and international recalls followed. Epidemiology-directed microbiological testing of suspect cheeses helped identify the outbreak source. Cross-contamination of cheese highlights the importance of using validated disinfectant protocols and routine cleaning and sanitizing after cutting each block or wheel.

Outbreak of Salmonella enterica serotype I 4,5,12:i:- infections: the challenges of hypothesis generation and microwave cooking.

We investigated an outbreak of 396 Salmonella enterica serotype I 4,5,12:i:- infections to determine the source. After 7 weeks of extensive hypothesis-generation interviews, no refined hypothesis was formed. Nevertheless, a case-control study was initiated. Subsequently, an iterative hypothesis-generation approach used by a single interviewing team identified brand A not-ready-to-eat frozen pot pies as a likely vehicle. The case-control study, modified to assess this new hypothesis, along with product testing indicated that the turkey variety of pot pies was responsible. Review of product labels identified inconsistent language regarding preparation, and the cooking instructions included undefined microwave wattage categories. Surveys found that most patients did not follow the product's cooking instructions and did not know their oven's wattage. The manufacturer voluntarily recalled pot pies and improved the product's cooking instructions. This investigation highlights the value of careful hypothesis-generation and the risks posed by frozen not-ready-to-eat microwavable foods.

Development and application of MLVA methods as a tool for inter-laboratory surveillance.

Multiple-locus variable-number of tandem-repeats analysis (MLVA) has emerged as a valuable method for subtyping bacterial pathogens and has been adopted in many countries as a critical component of their laboratory-based surveillance. Lack of harmonisation and standardisation of the method, however, has made comparison of results generated in different laboratories difficult, if not impossible, and has therefore hampered its use in international surveillance. This paper proposes an international consensus on the development, validation, nomenclature and quality control for MLVA used for molecular surveillance and outbreak detection based on a review of the current state of knowledge.

Four multistate outbreaks of human Salmonella infections associated with live poultry contact, United States, 2009.

Outbreaks of human salmonellosis associated with live poultry contact have been reported since 1955. Multiple Salmonella serotypes have been associated with these outbreaks, and specific outbreak strains have been repeatedly linked to single hatcheries over multiple years. During 2009, four multistate outbreaks of human Salmonella infections associated with direct and indirect exposure to live poultry purchased from mail-order hatcheries and agricultural feed stores were identified, resulting in 165 culture-confirmed cases in 30 states. This report describes the epidemiologic, environmental and laboratory investigations conducted by state and local health departments, state departments of agriculture, the U.S. Department of Agriculture (USDA), Animal and Plant Health Inspection Service (APHIS), National Poultry Improvement Plan (NPIP) and National Veterinary Services Laboratories (NVSL), and the Centers for Disease Control and Prevention (CDC). Case-patients were identified through PulseNet, the national molecular subtyping network for foodborne disease surveillance, and interviewed using the CDC standard live poultry contact questionnaire that asks about poultry-related exposures during the 7 days before illness onset. These outbreaks highlight the need to focus efforts on strategies to decrease and prevent human illness associated with live poultry contact through comprehensive interventions at the mail-order hatchery, agricultural feed store and consumer levels. Additional consumer education and interventions at mail-order hatcheries and venues where live poultry are sold, including agricultural feed stores, are necessary to prevent transmission of Salmonella from poultry to humans.

PulseNet USA: a five-year update.

PulseNet USA is the molecular surveillance network for foodborne infections in the United States. Since its inception in 1996, it has been instrumental in detection, investigation and control of numerous outbreaks caused by Shiga toxin-producing Escherichia coli O157:[H7] (STEC O157), Salmonella enterica, Listeria monocytogenes, Shigella spp., and Campylobacter. This paper describes the current status of the network, including the methodologies used and its future possibilities. The currently preferred subtyping method in the network is pulsed-field gel electrophoresis (PFGE), a proven highly discriminatory molecular subtyping method. New simpler sequencebased subtyping methods are under development and validation to complement and eventually replace PFGE. PulseNet is essentially a cluster detection network, but the data in the system will now also be used in attribution analyses of sporadic infections. The PulseNet platform will also be used as a primary tool in preparedness and response to acts of food bioterrorism.

Inhibition of growth of nonproteolytic Clostridium botulinum type B in sous vide cooked meat products is achieved by using thermal processing but not nisin.

The safety of refrigerated processed foods of extended durability (REPFEDs) with respect to nonproteolytic Clostridium botulinum is under continuous evaluation. In the present study, mild (P7.0(85.0) values 0 to 2 min [P, pasteurization value; z-value 7.0 degrees C; reference temperature 85.0 degrees C]) and increased (P7.0(85.0) values 67 to 515 min) heat treatments were evaluated in relation to survival of nonproteolytic C. botulinum type B spores in sous vide processed ground beef and pork cubes. The use of two concentrations of nisin in inhibition of growth and toxin production by nonproteolytic C. botulinum in the same products was also evaluated. A total of 96 samples were heat processed and analyzed for C. botulinum by BoNT/B gene-specific polmerase chain reaction and for botulinum toxin by a mouse bioassay after storage of 14 to 28 days at 4 and 8 degrees C. Predictably, after mild processing all samples of both products showed botulinal growth, and one ground beef sample became toxic at 8 degrees C. The increased heat processing, equivalent to 67 min at 85 degrees C. resulted in growth but not toxin production of C. botulinum in one ground beef sample in 21 days at 8 degrees C: in the pork cube samples no growth was detected. The increased heating of both products resulted in higher sensory quality than the milder heat treatment. Nisin did not inhibit the growth of nonproteolytic C. botulinum in either product; growth was detected in both products at 4 and 8 degrees C, and ground beef became toxic with all nisin levels within 21 to 28 days at 8 degrees C. Aerobic and lactic acid bacterial counts were reduced by the addition of nisin at 4 degrees C. The study demonstrates that the mild processing temperatures commonly employed in sous vide technology do not eliminate nonproteolytic C. botulinum type B spores. The intensity of each heat treatment needs to be carefully evaluated individually for each product to ensure product safety in relation to nonproteolytic C. botulinum.

Microbiological quality and shelf-life of vacuum-packaged 'gravad' rainbow trout stored at 3 and 8 degrees C.

Microbiological and sensory changes of vacuum-packaged 'gravad' rainbow trout slices were studied during storage at 3 and 8 degrees C. At the time of spoilage, after 27 and 20 days of storage at 3 and 8 degrees C, respectively, both mesophilic viable counts (MVC) and psychrotrophic viable counts (PVC) reached 10(6)-10(7) cfu/g at 3 degrees C and 10(7)-10(5) cfu/g at 8 degrees C. H2S-producing bacteria constituted a high proportion of the PVCs and lactic acid bacteria (LAB) counts were lower than the other determined bacterial counts. Sensory scores decreased with increasing MVC and PVC. The judges considered samples unfit for human consumption at MVC and PVC levels exceeding 10(6) and 10(7) cfu/g for samples stored at 3 and 8 degrees C, respectively. At respective levels of 10(7) and 10(8) cfu/g, most of the samples were deemed unfit. The main reasons for sensory rejection at both storage temperatures were the lack of the typical product odour or an ammonia off-odour and colour change to dark violet. The shelf-lives of the rainbow trout slices based on microbiological and sensory analyses were 20 days and 18 days at 3 and 8 degrees C, respectively.

Safety evaluation of sous vide-processed products with respect to nonproteolytic Clostridium botulinum by use of challenge studies and predictive microbiological models.

Sixteen different types of sous vide-processed products were evaluated for safety with respect to nonproteolytic group II Clostridium botulinum by using challenge tests with low (2. 0-log-CFU/kg) and high (5.3-log-CFU/kg) inocula and two currently available predictive microbiological models, Food MicroModel (FMM) and Pathogen Modeling Program (PMP). After thermal processing, the products were stored at 4 and 8 degrees C and examined for the presence of botulinal spores and neurotoxin on the sell-by date and 7 days after the sell-by date. Most of the thermal processes were found to be inadequate for eliminating spores, even in low-inoculum samples. Only 2 of the 16 products were found to be negative for botulinal spores and neurotoxin at both sampling times. Two products at the high inoculum level showed toxigenesis during storage at 8 degrees C, one of them at the sell-by date. The predictions generated by both the FMM thermal death model and the FMM and PMP growth models were found to be inconsistent with the observed results in a majority of the challenges. The inaccurate predictions were caused by the limited number and range of the controlling factors in the models. Based on this study, it was concluded that the safety of sous vide products needs to be carefully evaluated product by product. Time-temperature combinations used in thermal treatments should be reevaluated to increase the efficiency of processing, and the use of additional antibotulinal hurdles, such as biopreservatives, should be assessed.