Anti-LAMP-2 Antibody Seropositivity in Children with Primary Systemic Vasculitis Affecting Medium- and Large-Sized Vessels.

Chronic primary systemic vasculitis (PSV) comprises a group of heterogeneous diseases that are broadly classified by affected blood vessel size, clinical traits and the presence (or absence) of anti-neutrophil cytoplasmic antibodies (ANCA) against proteinase 3 (PR3) and myeloperoxidase (MPO). In small vessel vasculitis (SVV), ANCA are not present in all patients, and they are rarely detected in patients with vasculitis involving medium (MVV) and large (LVV) blood vessels. Some studies have demonstrated that lysosome-associated membrane protein-2 (LAMP-2/CD107b) is a target of ANCA in SVV, but its presence and prognostic value in childhood MVV and LVV is not known. This study utilized retrospective sera and clinical data obtained from 90 children and adolescents with chronic PSV affecting small (SVV, n = 53), medium (MVV, n = 16), and large (LVV, n = 21) blood vessels. LAMP-2-ANCA were measured in time-of-diagnosis sera using a custom electrochemiluminescence assay. The threshold for seropositivity was established in a comparator cohort of patients with systemic autoinflammatory disease. The proportion of LAMP-2-ANCA-seropositive individuals and sera concentrations of LAMP-2-ANCA were assessed for associations with overall and organ-specific disease activity at diagnosis and one-year follow up. This study demonstrated a greater time-of-diagnosis prevalence and sera concentration of LAMP-2-ANCA in MVV (52.9% seropositive) and LVV (76.2%) compared to SVV (45.3%). Further, LAMP-2-ANCA-seropositive individuals had significantly lower overall, but not organ-specific, disease activity at diagnosis. This did not, however, result in a greater reduction in disease activity or the likelihood of achieving inactive disease one-year after diagnosis. The results of this study demonstrate particularly high prevalence and concentration of LAMP-2-ANCA in chronic PSV that affects large blood vessels and is seronegative for traditional ANCA. Our findings invite reconsideration of roles for autoantigens other than MPO and PR3 in pediatric vasculitis, particularly in medium- and large-sized blood vessels.

From bytes to bites: Advancing the food industry with three-dimensional food printing.

The rapid advancement of three-dimensional (3D) printing (i.e., a type of additive manufacturing) technology has brought about significant advances in various industries, including the food industry. Among its many potential benefits, 3D food printing offers a promising solution to deliver products meeting the unique nutritional needs of diverse populations while also promoting sustainability within the food system. However, this is an emerging field, and there are several aspects to consider when planning for use of 3D food printing for large-scale food production. This comprehensive review explores the importance of food safety when using 3D printing to produce food products, including pathogens of concern, machine hygiene, and cleanability, as well as the role of macronutrients and storage conditions in microbial risks. Furthermore, postprocessing factors such as packaging, transportation, and dispensing of 3D-printed foods are discussed. Finally, this review delves into barriers of implementation of 3D food printers and presents both the limitations and opportunities of 3D food printing technology.

Transfer rates of Salmonella Typhimurium, Listeria monocytogenes, and a human norovirus surrogate impacted by macronutrient composition of food inks in 3D food printing systems.

3D food printers (3DFPs) allow for the customization of the physiochemical properties of foods in new ways. Transfer kinetics of foodborne pathogens between surfaces and food inks have not been evaluated in 3DFPs. This study aimed to determine if the macromolecular composition of food inks would impact the transfer rate of foodborne pathogens from the stainless steel food ink capsule to the 3D printed food. Salmonella Typhimurium, Listeria monocytogenes, and a human norovirus surrogate, Tulane virus (TuV), were inoculated onto the interior surface of stainless steel food ink capsules and dried for 30 min. Subsequently, 100 g of one of the following prepared food inks was extruded: (1) pure butter, (2) a powdered sugar solution, (3) a protein powder solution, and (4) a 1:1:1 ratio of all three macromolecules. Pathogen enumeration was completed for both the soiled capsules and the printed food products and resulting transfer rates were estimated using a generalized linear model with quasibinomial errors. A significant two-way interaction effect was found between microorganism type and food ink type (P = 0.0002). Tulane virus was typically transferred the most, and no significant differences between L. monocytogenes and S. Typhimurium were observed for any food matrix or across food matrices. Among food matrices, the complex mixture transferred fewer microorganisms in all instances, while butter, protein, and sugar were all statistically indistinguishable. This research seeks to further develop the field of 3DFP safety and to advance the understanding of the role of macromolecular composition in pathogen transfer kinetics, which have not previously been explored in pure matrices.

Factors Impacting Persistence of Phi6 Bacteriophage, an Enveloped Virus Surrogate, on Fomite Surfaces.

The persistence of Phi6 (Φ6) bacteriophage on surfaces commonly encountered in consumer-facing environments was evaluated. Φ6 has been utilized as a surrogate for enveloped viruses, including SARS-CoV-2-the causative agent of COVID-19-due to structural similarities, biosafety level 1 (BSL-1) status, and ease of use. Φ6 persistence on fomites was evaluated by characterizing the impact of the inoculum matrix (artificial saliva, phosphate-buffered saline [PBS], tripartite), inoculum level (low and high), and surface type (nonporous-aluminum, stainless steel, plastic, touchscreen, vinyl; porous-wood). Φ6 was inoculated onto surfaces at low and high inoculum levels for each inoculum matrix and incubated (20.54 ± 0.48°C) for up to 168 h. Φ6 was eluted from the surface and quantified via the double agar overlay assay to determine virus survival over time. For nonporous surfaces inoculated with artificial saliva and PBS, significantly higher D values were observed with high inoculum application according to the 95% confidence intervals. In artificial saliva, D values ranged from 1.00 to 1.35 h at a low inoculum and 4.44 to 7.05 h at a high inoculum across inoculation matrices and surfaces. D values for Φ6, regardless of the inoculum level, were significantly higher in tripartite than in artificial saliva and PBS for nonporous surfaces. In contrast with artificial saliva or PBS, D values in tripartite at low inoculum (D values ranging from 45.8 to 72.8 h) were greater than those at high inoculum (D values ranging from 26.4 to 45.5 h) on nonporous surfaces. This study characterized the impact of the inoculum matrix, inoculum level, and surface type on Φ6 survival on various surfaces relevant to fomite transmission in public settings. IMPORTANCE An important consideration in virus contact transmission is the transfer rate between hands and surfaces, which is driven by several factors, including virus persistence on inanimate surfaces. This research characterized Φ6 persistence on surfaces commonly encountered in public settings based on various factors. The inoculum matrix, which simulates the route of transmission, can impact virus persistence, and three separate matrices were evaluated in this study to determine the impact on Φ6 persistence over time. The number of microorganisms has also been suggested to impact persistence, which was evaluated here to simulate real-world contamination scenarios on six surface types. Results from this study will guide future research utilizing Φ6 or other surrogates for enveloped viruses of public health concern.

Transfer of Phi6 bacteriophage between human skin and surfaces common to consumer-facing environments.

AIMS: This study aimed to determine the extent of Phi6 (Φ6) transfer between skin and surfaces relevant to consumer-facing environments based on inoculum matrix, surface type and contact time. METHODS AND RESULTS: Φ6 transfer rates were determined from skin-to-fomite and fomite-to-skin influenced by inoculum matrix (artificial saliva and tripartite), surface type (aluminium, plastic, stainless steel, touchscreen, vinyl and wood) and contact time (5 and 10 s). Significant differences in estimated means were observed based on surface type (both transfer directions), inoculum matrix (skin-to-fomite) and contact time (both transfer directions). During a sequential transfer experiment from fomite-to-skin, the maximum number of consecutive transfer events observed was 3.33 ± 1.19, 2.33 ± 1.20 and 1.67 ± 1.21 for plastic, touchscreen and vinyl, respectively. CONCLUSIONS: Contact time significantly impacted Φ6 transfer rates, which may be attributed to skin absorption dynamics. Surface type should be considered for assessing Φ6 transfer rates. SIGNIFICANCE AND IMPACT OF THE STUDY: Although the persistence of Φ6 on fomites has been characterized, limited data are available regarding the transfer of Φ6 among skin and fomites. Determining Φ6 transfer rates for surfaces in consumer-facing environments based on these factors is needed to better inform future virus transmission mitigation strategies.

Pathogen and Surrogate Survival in Relation to Fecal Indicator Bacteria in Freshwater Mesocosms.

The microbial quality of agricultural water for fresh produce production is determined by the presence of the fecal indicator bacterium (FIB) Escherichia coli, despite poor correlations with pathogen presence. Additional FIB, such as enterococci, have been utilized for assessing water quality. The study objective was to determine the survival times (first time to detect zero or censored) of FIB (E. coli and enterococci), surrogates (Listeria innocua, Listeria seeligeri, Salmonella enterica serovar Typhimurium, and PRD1), and pathogens (four strains each of pathogenic E. coli and Listeria monocytogenes and five Salmonella serovars) simultaneously inoculated in freshwater mesocosms exposed to diel and seasonal variations. Six separate mesocosm experiments were conducted for ≤28 days each season, with samples (sediment/water) collected each day for the first 7 days and weekly thereafter. Microorganisms survived significantly longer in sediment than in water (hazard ratio [HR] for water/sediment is 2.2; 95% confidence interval [CI], 1.79 to 2.71). Also, FIB E. coli survived significantly longer than FIB enterococcus (HR for enterococci/E. coli is 12.9 [95% CI, 8.18 to 20.37]) after adjusting for the sediment/water and lake/river effects. Differences in the area under the curve (calculated from log CFU or PFU over time) were used to assess pathogen and surrogate survival in relation to FIB. Despite sample type (sediment/water) and seasonal influences, survival rates of pathogenic Salmonella serovars were similar to those of FIB E. coli, and survival rates of L. monocytogenes and pathogenic E. coli were similar to those of FIB enterococci. Further investigation of microbial survival in water and sediment is needed to determine which surrogates are best suited to assess pathogen survival in agricultural water used in irrigation water for fresh produce. IMPORTANCE Contamination of fresh produce via agricultural water is well established. This research demonstrates that survival of fecal indicator bacteria, pathogenic microorganisms, and other bacterial and viral surrogates in freshwater differs by sample type (sediment/water) and season. Our work highlights potential risks associated with pathogen accumulation and survival in sediment and the possibility for resuspension and contamination of agricultural water used in fresh produce production. Specifically, a greater microbial persistence in sediments than in water over time was observed, along with differences in survival among microorganisms in relation to the fecal indicator bacteria E. coli and enterococci. Previous studies compared data among microbial groups in different environments. Conversely, fecal indicator bacteria, surrogates, and pathogenic microorganisms were assessed within the same water and sediment mesocosms in the present study during four seasons, better representing the agricultural aquatic environment. These data should be considered when agricultural microbial water quality criteria in fresh produce operations are being determined.

Gut Microbiota Features on Nursing Home Admission Are Associated With Subsequent Acquisition of Antibiotic-resistant Organism Colonization.

Nursing home (NH) patients often acquire colonization with antibiotic-resistant organisms (AROs). We show that patients exposed to broad-spectrum antibiotics during previous hospitalizations have elevated enterococcal relative abundances on NH admission and higher risk of subsequent ARO acquisition. Our findings suggest that interventions preventing ARO spread should extend beyond NH doors.

Swabbing the surface: critical factors in environmental monitoring and a path towards standardization and improvement.

Cross-contamination can be broadly defined as the transfer, direct or indirect, of microorganisms from a contaminated product to a non-contaminated product. Events that may result in cross-contamination include inadequate hygiene practices, contaminated equipment surfaces, contamination via food handling personnel, further product processing, or storage abuse All of these niches require consistent environmental surveillance systems to monitor microbial harborage sites to prevent foodborne illnesses via cross-contamination. Environmental surveillance is achieved through routine surface sampling of the food contact surfaces and surrounding areas. To better understand cross-contamination, the role of environmental surface transmission during outbreaks due to the presence and persistence of pathogenic microorganisms on various food contact surfaces must be investigated. However, studies on environmental sampling techniques are rarely performed in an actual food processing environment but rather under controlled variables within a laboratory-setting. Moreover, results and conclusions of studies differ because of the considerable variability across surface sampling tools due to individual operator dependency, low recovery rates, and low reproducibility. Information is also often lacking on environmental sampling tools used within a processing facility, the characterization of these tools, and the optimization of recovery of microorganisms for surface sampling. Thus, this review aims to: (1) discuss and compare factors impacting the recovery of microorganisms and the standardization of surface sampling methods for optimal recovery of microorganisms and (2) examine how research strategies could focus more towards the development of standard methodologies for surface sampling.

Multidrug-resistant Organisms in Hospitals: What Is on Patient Hands and in Their Rooms?

BACKGROUND: The impact of healthcare personnel hand contamination in multidrug-resistant organism (MDRO) transmission is important and well studied; however, the role of patient hand contamination needs to be characterized further. METHODS: Patients from 2 hospitals in southeast Michigan were recruited within 24 hours of arrival to their room and followed prospectively using microbial surveillance of nares, dominant hand, and 6 high-touch environmental surfaces. Sampling was performed on admission, days 3 and 7, and weekly until discharge. Paired samples of methicillin-resistant Staphylococcus aureus (MRSA) isolated from the patients' hand and room surfaces were evaluated for relatedness using pulsed-field gel electrophoresis and staphylococcal cassette chromosome mec, and Panton-Valentine leukocidin typing. RESULTS: A total of 399 patients (mean age, 60.8 years; 49% male) were enrolled and followed for 710 visits. Fourteen percent (n = 56/399) of patients were colonized with an MDRO at baseline; 10% (40/399) had an MDRO on their hands. Twenty-nine percent of rooms harbored an MDRO. Six percent (14/225 patients with at least 2 visits) newly acquired an MDRO on their hands during their stay. New MDRO acquisition in patients occurred at a rate of 24.6/1000 patient-days, and in rooms at a rate of 58.6/1000 patient-days. Typing demonstrated a high correlation between MRSA on patient hands and room surfaces. CONCLUSIONS: Our data suggest that patient hand contamination with MDROs is common and correlates with contamination on high-touch room surfaces. Patient hand hygiene protocols should be considered to reduce transmission of pathogens and healthcare-associated infections.

Virus-Bacteria Interactions: Implications for Prevention and Control of Human Enteric Viruses from Environment to Host.

Human enteric viruses, specifically human norovirus (hNoV), are the most common cause of foodborne illness boasting a wide range of transmission routes. These include person to person, contact with contaminated fomites, as well as ingestion of contaminated water and food. Because of this, the control and prevention of enteric viruses in food and other relevant environments have been a research focus over the past few decades. Interestingly, viruses as well as many other pathogens are often studied in isolation even though it is known that microorganisms do not occur in isolation but rather as part of complex microbial communities-both external from the host and within the host. Therefore, the overall goal of this review is to present the current evidence on virus-microbe interactions as these relate to the infectivity as well as the control and prevention of epidemiologically relevant foodborne viruses (such as hNoV) within our food systems. Therefore, this review is divided into in vivo, in situ, and in vitro implications of virus-microbe interactions through discussion of studies investigating the complex relationships between human enteric viruses and microbial cohabitants, specifically hNoV and bacteria.

Longitudinal Assessment of Multidrug-Resistant Organisms in Newly Admitted Nursing Facility Patients: Implications for an Evolving Population.

Background: The spread of multidrug-resistant organisms (MDROs) is a global concern, and much about transmission in healthcare systems remains unknown. To reduce hospital stays, nursing facilities (NFs) have increasingly assumed care of post-acute populations. We estimate the prevalence of MDRO colonization in NF patients on enrollment and discharge to community settings, risk factors for colonization, and rates of acquiring MDROs during the stay. Methods: We conducted a prospective, longitudinal cohort study of newly admitted patients in 6 NFs in southeast Michigan using active microbial surveillance of multiple anatomic sites sampled at enrollment, days 14 and 30, and monthly thereafter for up to 6 months. Results: We enrolled 651 patients and collected 7526 samples over 1629 visits, with an average of 29 days of follow-up per participant. Nearly all participants were admitted for post-acute care (95%). More than half (56.8%) were colonized with MDROs at enrollment: methicillin-resistant Staphylococcus aureus (MRSA), 16.1%; vancomycin-resistant enterococci (VRE), 33.2%; and resistant gram-negative bacilli (R-GNB), 32.0%. Risk factors for colonization at enrollment included prolonged hospitalization (>14 days), functional disability, antibiotic use, or device use. Rates per 1000 patient-days of acquiring a new MDRO were MRSA, 3.4; VRE, 8.2; and R-GNB, 13.6. MDRO colonization at discharge was similar to that at enrollment (56.4%): MRSA, 18.4%; VRE, 30.3%; and R-GNB, 33.6%. Conclusions: Short-stay NF patients exhibit a high prevalence of MDROs near the time of admission, as well as at discharge, and may serve as a reservoir for spread in other healthcare settings. Future interventions to reduce MDROs should specifically target this population.

Environmental Panels as a Proxy for Nursing Facility Patients With Methicillin-Resistant Staphylococcus aureus and Vancomycin-Resistant Enterococcus Colonization.

Background: Most nursing facilities (NFs) lack methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) surveillance programs due to limited resources and high costs. We investigated the utility of environmental screening of high-touch surfaces in patient rooms as a way to circumvent these challenges. Methods: We compared MRSA and VRE culture data from high-touch surfaces in patients' rooms (14450 samples from 6 NFs) and ranked each site's performance in predicting patient colonization (7413 samples). The best-performing sites were included in a MRSA- and a VRE-specific panel that functioned as a proxy for patient colonization. Molecular typing was performed to confirm available concordant patient-environment pairs. Results: We identified and validated a MRSA panel that consisted of the bed controls, nurse call button, bed rail, and TV remote control. The VRE panel included the toilet seat, bed controls, bed rail, TV remote control, and top of the side table. Panel colonization data tracked patient colonization. Negative predictive values were 89%-92% for MRSA and 82%-84% for VRE. Molecular typing confirmed a strong clonal type relationship in available concordant patient-environment pairs (98% for MRSA, 91% for VRE), pointing to common epidemiological patterns for environmental and patient isolates. Conclusions: Environmental panels used as a proxy for patient colonization and incorporated into facility surveillance protocols can guide decolonization strategies, improve awareness of MRSA and VRE burden, and inform efforts to reduce transmission. Targeted environmental screening may be a viable surveillance strategy for MRSA and VRE detection in NFs.

Identification of Factors Affecting Fecal Pollution in Beaver Lake Reservoir.

Standard methods for the evaluation of recreational water quality rely on generic bacterial indicators such as . However, does not provide enough information to determine fecal source or public health risk. The stsudy objective was to determine factors influencing the presence of and host-specific markers (HSM) from upstream to downstream in Beaver Lake Reservoir (BLR). From February 2014 to September 2015, 420 base flow and rain event samples were collected from seven sites-two sites from streams (White River [WR] and War Eagle Creek) draining into BLR and five sites from within BLR. Each sample was analyzed for and by quantitative polymerase chain reaction for HSM related to human, bovine, and poultry. The data indicate that overall levels of were significantly greater in the WR and significantly lower at the most downstream sampling location in BLR. is more likely present during spring (adjusted odds ratio [aOR] = 1.86), at the WR sampling site (aOR = 3.39), or during a rain event (aOR = 2.73). Moreover, the HSM HumM2 is more likely present (aOR = 1.99) when is present. These same factors were associated with concentrations >126 most probable number 100 mL (aOR = 2.76-12.48), except the poultry marker CL was more likely associated (aOR = 3.81) than HumM2. This study revealed that both seasonal and locational factors are important variables for fecal pollution in BLR. Moreover, these same factors may apply to fecal pollution in manmade reservoirs within similar types of watersheds across the United States, as well as internationally.

Environmental persistence of Tulane virus - a surrogate for human norovirus.

Human noroviruses (HuNoVs) are the leading cause of acute viral gastroenteritis worldwide. The persistence of HuNoV in the environment contributes significantly to its transmission to humans. Surrogate viruses are used to study HuNoV owing to the lack of a cell culture system for this virus. Here, the persistence of Tulane virus (TV) - a novel HuNoV surrogate - in surface water (SW) and groundwater (GW) as well as on acrylic-based solid (ABS) and stainless steel (SS) surfaces was investigated. After 28 days, TV remained stable in SW (<1 log10 reduction) but was reduced by ≥3.5 to 4 log10 in GW by day 21. TV had a higher rate of reduction on SS compared with ABS, with corresponding D values of 18.5 ± 0.34 and 13.1 ± 0.36 days, respectively. This is the first study to demonstrate the persistence of TV in environmental waters and on fomite surfaces.

Combined Application of Essential Oil Compounds and Bacteriophage to Inhibit Growth of Staphylococcus aureus In Vitro.

Staphylococcus aureus is considered an important human pathogen. This study aimed to investigate the combination of essential oil compounds (EOCs) and bacteriophage as alternative antimicrobials to control S. aureus in vitro. Here, four EOCs (alpha-pinene, 3-carene, (+)-limonene, (1S)-(-)-ß-pinene) were evaluated by disc diffusion assay (DDA) and growth inhibition assay (GIA) to determine inhibitory effects against five strains of S. aureus. Phage adsorption assays were performed with phage K up to 120 h at 6, 13, and 37 °C to determine lytic activity. Combinations of phage K and EOCs against S. aureus were also evaluated at 37 °C. Alpha-pinene exhibited significantly greater inhibition towards S. aureus strains when compared to other EOCs tested by the DDA. GIAs indicate that all S. aureus strains exhibited significantly reduced growth (P < 0.006) over a 48-h period when exposed to EOCs. Phage adsorption assays indicate that phage K has high lytic activity at 37 °C with at least a 1.5-log increase in the number of plaque-forming units (PFU) over 6 h when compared to 6 and 13 °C. S. aureus strains showed significantly reduced growth (P < 0.05) when treated with combined phage K and EOCs. Results from the combined effect of EOC and phage indicate that phage alone inhibits S. aureus in vitro at 37 °C as effectively as EOCs alone or in combination with phage with variability between strains. The results from DDA, GIA, and phage adsorption assays indicate that select EOCs and phage K can be used as antimicrobials against S. aureus in vitro with potential application in situ.

Interactions between Human Norovirus Surrogates and Acanthamoeba spp.

Human noroviruses (HuNoVs) are the most common cause of food-borne disease outbreaks, as well as virus-related waterborne disease outbreaks in the United States. Here, we hypothesize that common free-living amoebae (FLA)-ubiquitous in the environment, known to interact with pathogens, and frequently isolated from water and fresh produce-could potentially act as reservoirs of HuNoV and facilitate the environmental transmission of HuNoVs. To investigate FLA as reservoirs for HuNoV, the interactions between two Acanthamoeba species, A. castellanii and A. polyphaga, as well as two HuNoV surrogates, murine norovirus type 1 (MNV-1) and feline calicivirus (FCV), were evaluated. The results showed that after 1 h of amoeba-virus incubation at 25°C, 490 and 337 PFU of MNV-1/ml were recovered from A. castellanii and A. polyphaga, respectively, while only few or no FCVs were detected. In addition, prolonged interaction of MNV-1 with amoebae was investigated for a period of 8 days, and MNV-1 was demonstrated to remain stable at around 200 PFU/ml from day 2 to day 8 after virus inoculation in A. castellanii. Moreover, after a complete amoeba life cycle (i.e., encystment and excystment), infectious viruses could still be detected. To determine the location of virus associated with amoebae, immunofluorescence experiments were performed and showed MNV-1 transitioning from the amoeba surface to inside the amoeba over a 24-h period. These results are significant to the understanding of how HuNoVs may interact with other microorganisms in the environment in order to aid in its persistence and survival, as well as potential transmission in water and to vulnerable food products such as fresh produce.

Genes Indicative of Zoonotic and Swine Pathogens Are Persistent in Stream Water and Sediment following a Swine Manure Spill.

Manure spills into streams are relatively frequent, but no studies have characterized stream contamination with zoonotic and veterinary pathogens, or fecal chemicals, following a spill. We tested stream water and sediment over 25 days and downstream for 7.6 km for the following: fecal indicator bacteria (FIB), the fecal indicator chemicals cholesterol and coprostanol, 20 genes for zoonotic and swine-specific bacterial pathogens by presence/absence PCR for viable cells, one swine-specific Escherichia coli toxin gene (STII gene) by quantitative PCR (qPCR), and nine human and animal viruses by qPCR or reverse transcription-qPCR. Twelve days postspill, and 4.2 km downstream, water concentrations of FIB, cholesterol, and coprostanol were 1 to 2 orders of magnitude greater than those detected before, or above, the spill, and genes indicating viable zoonotic or swine-infectious Escherichia coli were detected in water or sediment. STII gene levels increased from undetectable before or above the spill to 10(5) copies/100 ml of water 12 days postspill. Thirteen of 14 water (8/9 sediment) samples had viable STII-carrying cells postspill. Eighteen days postspill, porcine adenovirus and teschovirus were detected 5.6 km downstream. FIB concentrations (per gram [wet weight]) in sediment were greater than in water, and sediment was a continuous reservoir of genes and chemicals postspill. Constituent concentrations were much lower, and detections less frequent, in a runoff event (200 days postspill) following manure application, although the swine-associated STII and stx2e genes were detected. Manure spills are an underappreciated pathway for livestock-derived contaminants to enter streams, with persistent environmental outcomes and the potential for human and veterinary health consequences.

A comparison of two informative SNP-based strategies for typing Pseudomonas aeruginosa isolates from patients with cystic fibrosis.

BACKGROUND: Molecular typing is integral for identifying Pseudomonas aeruginosa strains that may be shared between patients with cystic fibrosis (CF). We conducted a side-by-side comparison of two P. aeruginosa genotyping methods utilising informative-single nucleotide polymorphism (SNP) methods; one targeting 10 P. aeruginosa SNPs and using real-time polymerase chain reaction technology (HRM10SNP) and the other targeting 20 SNPs and based on the Sequenom MassARRAY platform (iPLEX20SNP). METHODS: An in-silico analysis of the 20 SNPs used for the iPLEX20SNP method was initially conducted using sequence type (ST) data on the P. aeruginosa PubMLST website. A total of 506 clinical isolates collected from patients attending 11 CF centres throughout Australia were then tested by both the HRM10SNP and iPLEX20SNP assays. Type-ability and discriminatory power of the methods, as well as their ability to identify commonly shared P. aeruginosa strains, were compared. RESULTS: The in-silico analyses showed that the 1401 STs available on the PubMLST website could be divided into 927 different 20-SNP profiles (D-value = 0.999), and that most STs of national or international importance in CF could be distinguished either individually or as belonging to closely related single- or double-locus variant groups. When applied to the 506 clinical isolates, the iPLEX20SNP provided better discrimination over the HRM10SNP method with 147 different 20-SNP and 92 different 10-SNP profiles observed, respectively. For detecting the three most commonly shared Australian P. aeruginosa strains AUST-01, AUST-02 and AUST-06, the two methods were in agreement for 80/81 (98.8%), 48/49 (97.8%) and 11/12 (91.7%) isolates, respectively. CONCLUSIONS: The iPLEX20SNP is a superior new method for broader SNP-based MLST-style investigations of P. aeruginosa. However, because of convenience and availability, the HRM10SNP method remains better suited for clinical microbiology laboratories that only utilise real-time PCR technology and where the main interest is detection of the most highly-prevalent P. aeruginosa CF strains within Australian clinics.

Tulane Virus Persistence and Microbial Stability in 3D Food Ink under Various Storage Conditions: A Pre- and Post-Printing Analysis.

3D food printers facilitate novel customization of the physicochemical properties of food. This study aimed to investigate the impact of storage conditions on the inactivation of the human norovirus surrogate, Tulane virus (TuV), within 3D printed foods. TuV-inoculated protein cookie food ink (∽ 4 log PFU/g) was distributed into 18 3D food printer capsules (50 g each); half immediately underwent extrusion. Storage of the capsules and printed food products at 20 °C (0, 6, 12, and 24 h), 4 °C (0, 1, 3, and 5d), and - 18 °C (0, 1, 3, and 5d) was completed before analysis for TuV via plaque assays in addition to aerobic plate count, yeast and mold counts, and pH and water activity (aw) measurements. A significant 3-way interaction effect was observed between time, temperature, and storage method (capsule/print) (p = 0.006). Significant findings include: (1) A greater reduction in virions was observed in capsules after 24 h at 20 °C and (2) a substantial reduction in virions at 4 °C from day 0 to day 1 was observed, independent of storage method. Microbial indicators remained steady across temperatures, with storage temperature significantly impacting pH and aw. A significant two-way interaction effect (p = 0.006) was found between microorganism type (yeast/aerobic counts) and temperature. This research seeks to provide insights for the food industry and regulatory bodies in crafting guidelines for the safe storage and handling of 3D printed foods and inks.

Hydroponic Nutrient Solution Temperature Impacts Tulane Virus Persistence over Time.

Controlled environment agriculture (CEA), or indoor agriculture, encompasses non-traditional farming methods that occur inside climate-controlled structures (e.g., greenhouses, warehouses, high tunnels) allowing for year-round production of fresh produce such as leaf lettuce. However, recent outbreaks and recalls associated with hydroponically grown lettuce contaminated with human pathogens have raised concerns. Few studies exist on the food safety risks during hydroponic cultivation of leaf lettuce; thus, it is important to identify contributing risk factors and potential mitigation strategies to prevent foodborne transmission via hydroponically grown produce. In this study, the concentration of infectious Tulane virus (TV), a human norovirus surrogate, in hydroponic nutrient solution at 15 °C, 25 °C, 30 °C, and 37 °C was determined over a duration of 21 days to mimic the time from seedling to mature lettuce. The mean log PFU reduction for TV was 0.86, 1.80, 2.87, and ≥ 3.77 log10 at 15 °C, 25 °C, 30 °C, and 37 °C, respectively, at the end of the 21-day period. Similarly, average decimal reduction values (D-values) of TV at 15 °C, 25 °C, 30 °C, and 37 °C were 48.0, 11.3, 8.57, and 7.02 days, respectively. This study aids in the (i) identification of possible food safety risks associated with hydroponic systems specifically related to nutrient solution temperature and (ii) generation of data to perform risk assessments within CEA leaf lettuce operations to inform risk management strategies for the reduction of foodborne outbreaks, fresh produce recalls, and economic losses.