Interlaboratory Evaluation of Enterococcus faecium NRRL B-2354 as a Salmonella Surrogate for Validating Thermal Treatment of Multiple Low-Moisture Foods.

ABSTRACT: This multi-institutional study assessed the efficacy of Enterococcus faecium NRRL B-2354 as a nonpathogenic Salmonella surrogate for thermal processing of nonfat dry milk powder, peanut butter, almond meal, wheat flour, ground black pepper, and date paste. Each product was analyzed by two laboratories (five independent laboratories total), with the lead laboratory inoculating (E. faecium or a five-strain Salmonella enterica serovar cocktail of Agona, Reading, Tennessee, Mbandaka, and Montevideo) and equilibrating the product to the target water activity before shipping. Both laboratories subjected samples to three isothermal treatments (between 65 and 100°C). A log-linear and Bigelow model was fit to survivor data via one-step regression. On the basis of D80°C values estimated from the combined model, E. faecium was more thermally resistant (P < 0.05) than Salmonella in nonfat dry milk powder (DEf-80°C, 100.2 ± 5.8 min; DSal-80°C, 28.9 ± 1.0 min), peanut butter (DEf-80°C, 133.5 ± 3.1 min; DSal-80°C, 57.6 ± 1.5 min), almond meal (DEf-80°C, 34.2 ± 0.4 min; DSal-80°C, 26.1 ± 0.2 min), ground black pepper (DEf-80°C, 3.2 ± 0.8 min; DSal-80°C, 1.5 ± 0.1 min), and date paste (DEf-80°C, 1.5 ± 0.0 min; DSal-80°C, 0.5 ± 0.0 min). Although the combined laboratory D80°C for E. faecium was lower (P < 0.05) than for Salmonella in wheat flour (DEf-80°C, 9.4 ± 0.1 min; DSal-80°C, 10.1 ± 0.2 min), the difference was ∼7%. The zT values for Salmonella in all products and for E. faecium in milk powder, almond meal, and date paste were not different (P > 0.05) between laboratories. Therefore, this study demonstrated the impact of standardized methodologies on repeatability of microbial inactivation results. Overall, E. faecium NRRL B-2354 was more thermally resistant than Salmonella, which provides support for utilizing E. faecium as a surrogate for validating thermal processing of multiple low-moisture products. However, product composition should always be considered before making that decision.

Interlaboratory comparison of SARS-CoV2 molecular detection assays in use by U.S. veterinary diagnostic laboratories.

The continued search for intermediate hosts and potential reservoirs for SARS-CoV2 makes it clear that animal surveillance is critical in outbreak response and prevention. Real-time RT-PCR assays for SARS-CoV2 detection can easily be adapted to different host species. U.S. veterinary diagnostic laboratories have used the CDC assays or other national reference laboratory methods to test animal samples. However, these methods have only been evaluated using internal validation protocols. To help the laboratories evaluate their SARS-CoV2 test methods, an interlaboratory comparison (ILC) was performed in collaboration with multiple organizations. Forty-four sets of 19 blind-coded RNA samples in Tris-EDTA (TE) buffer or PrimeStore transport medium were shipped to 42 laboratories. Results were analyzed according to the principles of the International Organization for Standardization (ISO) 16140-2:2016 standard. Qualitative assessment of PrimeStore samples revealed that, in approximately two-thirds of the laboratories, the limit of detection with a probability of 0.95 (LOD95) for detecting the RNA was ≤20 copies per PCR reaction, close to the theoretical LOD of 3 copies per reaction. This level of sensitivity is not expected in clinical samples because of additional factors, such as sample collection, transport, and extraction of RNA from the clinical matrix. Quantitative assessment of Ct values indicated that reproducibility standard deviations for testing the RNA with assays reported as N1 were slightly lower than those for N2, and they were higher for the RNA in PrimeStore medium than those in TE buffer. Analyst experience and the use of either a singleplex or multiplex PCR also affected the quantitative ILC test results.

Thermal Inactivation of Salmonella Agona in Low-Water Activity Foods: Predictive Models for the Combined Effect of Temperature, Water Activity, and Food Component.

Salmonella can survive in low-moisture, high-protein, and high-fat foods for several years. Despite nationwide outbreaks and recalls due to the presence of Salmonella in low-moisture foods, information on thermal inactivation of Salmonella in these products is limited. This project evaluated the impact of water activity (aw), temperature, and food composition on thermal inactivation of Salmonella enterica serovar Agona in defined high-protein and high-fat model food matrices. Each matrix was inoculated with Salmonella Agona and adjusted to obtain a target aw, ranging from 0.50 to 0.98. Samples were packed into aluminum test cells and heated (52 to 90°C) under isothermal conditions. Survival of Salmonella Agona was detected on tryptic soy agar with 0.6% yeast extract. Complex influences by food composition, aw, and temperature resulted in significantly different ( P < 0.05) thermal resistance of Salmonella for the conditions tested. It was estimated that the same point temperatures at which the D-values of the two matrices at each aw (0.63, 0.73, 0.81, and 0.90) were identical were 79.48, 71.28, 69.62, and 38.42°C, respectively. Above these temperatures, the D-values in high-protein matrices were larger than the D-values in high-fat matrices at each aw. Below these temperatures, the inverse relationship was observed. A correlation between temperature and aw existed on the basis of the level of fat or protein in the food, showing that these compositional factors must be accounted for when predicating thermal inactivation of Salmonella in foods.

Salmonella Inactivation During Extrusion of an Oat Flour Model Food.

Little research exists on Salmonella inactivation during extrusion processing, yet many outbreaks associated with low water activity foods since 2006 were linked to extruded foods. The aim of this research was to study Salmonella inactivation during extrusion of a model cereal product. Oat flour was inoculated with Salmonella enterica serovar Agona, an outbreak strain isolated from puffed cereals, and processed using a single-screw extruder at a feed rate of 75 kg/h and a screw speed of 500 rpm. Extrudate samples were collected from the barrel outlet in sterile bags and immediately cooled in an ice-water bath. Populations were determined using standard plate count methods or a modified most probable number when populations were low. Reductions in population were determined and analyzed using a general linear model. The regression model obtained for the response surface tested was Log (NR /NO ) = 20.50 + 0.82T - 141.16aw - 0.0039T2 + 87.91aw2 (R2 = 0.69). The model showed significant (p < 0.05) linear and quadratic effects of aw and temperature and enabled an assessment of critical control parameters. Reductions of 0.67 ± 0.14 to 7.34 ± 0.02 log CFU/g were observed over ranges of aw (0.72 to 0.96) and temperature (65 to 100 °C) tested. Processing conditions above 82 °C and 0.89 aw achieved on average greater than a 5-log reduction of Salmonella. Results indicate that extrusion is an effective means for reducing Salmonella as most processes commonly employed to produce cereals and other low water activity foods exceed these parameters. Thus, contamination of an extruded food product would most likely occur postprocessing as a result of environmental contamination or through the addition of coatings and flavorings.

Survival of Salmonella on chamomile, peppermint, and green tea during storage and subsequent survival or growth following tea brewing.

The survival of Salmonella on dried chamomile flowers, peppermint leaves, and green tea leaves stored under different conditions was examined. Survival and growth of Salmonella was also assessed after subsequent brewing using dried inoculated teas. A Salmonella enterica serovar cocktail was inoculated onto different dried tea leaves or flowers to give starting populations of approximately 10 log CFU/g. The inoculum was allowed to dry (at ambient temperature for 24 h) onto the dried leaves or flowers prior to storage under 25 and 35 °C at low (<30% relative humidity [RH]) and high (>90% RH) humidity levels. Under the four storage conditions tested, survival followed the order 25 °C with low RH > 35 °C with low RH > 25 °C with high RH > 35 °C with high RH. Salmonella losses at 25 °C with low RH occurred primarily during drying, after which populations showed little decline over 6 months. In contrast, Salmonella decreased below detection after 45 days at 35 °C and high RH in all teas tested. The thermal resistance of Salmonella was assessed at 55 °C immediately after inoculation of tea leaves or flowers, after drying (24 h) onto tea leaves or flowers, and after 28 days of storage at 25 °C with low RH. All conditions resulted in similar D-values (2.78 ± 0.12, 3.04 ± 0.07, and 2.78 ± 0.56, at 0 h, 24 h, and 28 days, respectively), indicating thermal resistance of Salmonella in brewed tea did not change after desiccation and 28 days of storage. In addition, all brewed teas tested supported the growth of Salmonella. If Salmonella survives after storage, it may also survive and grow after a home brewing process.

Multicellular bacteria deploy the type VI secretion system to preemptively strike neighboring cells.

The Type VI Secretion System (T6SS) functions in bacteria as a contractile nanomachine that punctures and delivers lethal effectors to a target cell. Virtually nothing is known about the lifestyle or physiology that dictates when bacteria normally produce their T6SS, which prevents a clear understanding of how bacteria benefit from its action in their natural habitat. Proteus mirabilis undergoes a characteristic developmental process to coordinate a multicellular swarming behavior and will discriminate itself from another Proteus isolate during swarming, resulting in a visible boundary termed a Dienes line. Using transposon mutagenesis, we discovered that this recognition phenomenon requires the lethal action of the T6SS. All mutants identified in the genetic screen had insertions within a single 33.5-kb region that encodes a T6SS and cognate Hcp-VrgG-linked effectors. The identified T6SS and primary effector operons were characterized by killing assays, by construction of additional mutants, by complementation, and by examining the activity of the type VI secretion system in real-time using live-cell microscopy on opposing swarms. We show that lethal T6SS-dependent activity occurs when a dominant strain infiltrates deeply beyond the boundary of the two swarms. Using this multicellular model, we found that social recognition in bacteria, underlying killing, and immunity to killing all require cell-cell contact, can be assigned to specific genes, and are dependent on the T6SS. The ability to survive a lethal T6SS attack equates to "recognition". In contrast to the current model of T6SS being an offensive or defensive weapon our findings support a preemptive mechanism by which an entire population indiscriminately uses the T6SS for contact-dependent delivery of effectors during its cooperative mode of growth.

Eruptive xanthoma: a case report.

Eruptive xanthomatosis is a papular skin disorder resulting from hyperlipidemia, specifically hypertriglyceridemia. It is characterized by yellowish red papules concentrated on extensor surfaces of the arms and legs. The hyperlipidemia responsible for this disorder can be caused by a primary genetic defect, a secondary disorder, or both. Eruptive xanthomas often rapidly resolve after treatment of the hyperlipidemia has begun.