Efficacy of Three Antimicrobials Against two SARS-COV-2 Surrogates, Bovine Coronavirus and Human Coronavirus OC43, on Hard or Soft Nonporous Materials.

The efficacy of three antimicrobials was evaluated against two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surrogates - bovine coronavirus (BCoV) and human coronavirus (HCoV) OC43 - on hard and soft nonporous materials. Three antimicrobials with three different active ingredients (chlorine, hydrogen peroxide, and quaternary ammonium compound + alcohol) were studied. Initially, a neutralization method was optimized for each antimicrobial. Then, we determined their efficacy against BCoV and HCoV OC43 in both suspension and on surfaces made with polyethylene terephthalate (PET) plastic and vinyl upholstery fabric. All tests were conducted under ambient environmental conditions with a soil load of 5% fetal bovine serum. After a 2-min exposure, all three antimicrobials achieved a >3.0 log10 reduction in viral titers in suspension. All three also reduced virus infectivity on both surface materials below the detection limit (0.6 log10 TCID50/carrier). Treatments in which the reduction in virus titer was <3.0 log10 were attributed to a decreased dynamic range on the carrier during drying prior to disinfection. The carrier data revealed that both surrogates were inactivated more rapidly (p <0.05) on vinyl or under conditions of high relative humidity. Three classes of antimicrobials were efficacious against both SARS-CoV-2 surrogate viruses, with BCoV demonstrating slightly less sensitivity compared to HCoV OC43. These findings also illustrate the importance of (1) optimizing the neutralization method and (2) considering relative humidity as a key factor for efficacy testing.

Biological Control of Escherichia coli O157:H7 in Dairy Manure-Based Compost Using Competitive Exclusion Microorganisms.

BACKGROUND: Animal manure-based compost is a valuable organic fertilizer and biological soil amendment. To ensure the microbiological safety of compost products, the effectiveness of competitive exclusion microorganisms (CE) in reducing Escherichia coli O157:H7 in dairy manure-based compost was evaluated. METHODS: A cocktail of E. coli O157:H7 strains were inoculated into dairy compost along with CE strains isolated from compost, and the reduction in E. coli O157:H7 by CE was determined in compost with 20%, 30%, and 40% moisture levels at 22 °C and 30 °C under laboratory conditions, as well as in fall, winter, and summer seasons under greenhouse settings. RESULTS: Under lab conditions, CE addition resulted in 1.1-3.36 log reductions in E. coli O157:H7 in compost, with enhanced pathogen reduction by higher moisture and lower temperature. In the greenhouse, >99% of the E. coli O157:H7 population in compost with ≥30% moisture due to cross-contamination can be effectively inactivated by CE within 2 days during colder seasons. However, it took ≥8 days to achieve the same level of reduction for heat-adapted E. coli O157:H7 cells. CONCLUSIONS: Our results demonstrated that the competitive exclusion of microorganisms can be an effective tool for controlling foodborne pathogens in compost and reducing the potential for soil and crop contamination.

Efficacy of three EPA-registered antimicrobials and steam against two human norovirus surrogates on nylon carpets with two backing types.

Carpet cleaning guidelines currently do not include the use of an antimicrobial, except after a bodily fluid event. To address this gap, we compared the efficacy of three antimicrobials-two hydrogen peroxide-based (H2O2) products (A and B) and one chlorine-based product (C)-and a steam treatment against two norovirus surrogates, specifically feline calicivirus (FCV) and Tulane virus (TuV). These tests were performed on nylon carpets with either water-permeable or waterproof backing types. The effect of repeated antimicrobial use on carpet properties was also evaluated. For a carpet with water-permeable backing, products A, B, and C achieved a 0.8, 3.1, and 0.9 log10 PFU/coupon reduction of FCV and 0.3, 2.5, and 0.4 log10 TCID50/coupon reduction of TuV, respectively, following a 30 min contact time. For carpet with waterproof backing, only product B achieved a 5.0 log10 PFU/coupon reduction of FCV and >3.0 log10 TCID50/coupon reduction of TuV, whereas products A and C achieved a 2.4 and 1.6 log10 PFU/coupon reduction of FCV and a 1.2 and 1.2 log10 TCID50/coupon reduction of TuV, respectively. Steam treatment achieved a ≥ 5.2 log10 PFU/coupon reduction of FCV and a > 3.2 log10 TCID50/coupon reduction of TuV in 15 seconds on the carpet with both backing types. The repeated use of products A and B decreased the tensile strength of the carpet backing, while use of product B resulted in cracks on carpet fibers. Overall, steam treatment for 15 seconds was efficacious on both carpet types, but only product B achieved efficacy after a 30-minute exposure on the carpet with waterproof backing.IMPORTANCECarpets are common in long-term care facilities, despite its potential as a vehicle for transmission of agents associated with healthcare-associated infections, including human norovirus (NoV). Presently, our understanding of carpet disinfection is limited; hence, there are no commercial antimicrobials against norovirus available for use on carpets. Our findings showed that steam treatment, which minimally affected the properties of carpet fibers and backing, was more efficacious against human norovirus surrogates on carpets compared to the three chemical antimicrobials tested. Additionally, the two surrogates were more sensitive to chemical antimicrobials on the carpet with waterproof backing compared to carpets with water-permeable backing. These findings can inform development of antimicrobials for use on carpets contaminated with human norovirus.

Vapor-induced phase-separation-enabled versatile direct ink writing.

Versatile printing of polymers, metals, and composites always calls for simple, economic approaches. Here we present an approach to three-dimensional (3D) printing of polymeric, metallic, and composite materials at room conditions, based on the polymeric vapor-induced phase separation (VIPS) process. During VIPS 3D printing (VIPS-3DP), a dissolved polymer-based ink is deposited in an environment where nebulized non-solvent is present, inducing the low-volatility solvent to be extracted from the filament in a controllable manner due to its higher chemical affinity with the non-solvent used. The polymeric phase is hardened in situ as a result of the induced phase separation process. The low volatility of the solvent enables its reclamation after the printing process, significantly reducing its environmental footprint. We first demonstrate the use of VIPS-3DP for polymer printing, showcasing its potential in printing intricate structures. We further extend VIPS-3DP to the deposition of polymer-based metallic inks or composite powder-laden polymeric inks, which become metallic parts or composites after a thermal cycle is applied. Furthermore, spatially tunable porous structures and functionally graded parts are printed by using the printing path to set the inter-filament porosity as well as an inorganic space-holder as an intra-filament porogen.

Nigella sativa as an antibiotic alternative to promote growth and enhance health of broilers challenged with Eimeria maxima and Clostridium perfringens.

The poultry industry has significant coccidiosis and necrotic enteritis (NE) challenges, leading to high mortality and unacceptable growth without antibiotic treatment. This research explored supplementing Nigella sativa (black cumin) seed oil in poultry feed to mitigate coccidiosis and prevent or lessen NE in broilers. In vivo studies consisted of 384 and 320 Cobb 500 male broiler chicks distributed in a randomized complete block experimental design for trials 1 and 2, respectively. The first trial compared 3 concentrations (1, 2, and 5 mL/kg) of black cumin seed oil (BCSO), and trial 2 compared 2 concentrations (2 and 5 mL/kg) BCSO, with birds challenged with Eimeria maxima and Clostridium perfringens (Cp) strains Cp#6 and Cp#4, respectively. Broiler live performance, NE disease outcomes, and Cp populations were measured for both trials. A commercially available BCSO oil product, determined in a preliminary in vitro study to have the highest anti-Cp activity, was selected for in vivo studies. Gas chromatography-mass spectrometry analysis indicated the major bioactive compounds p-cymene, thymoquinone, carvacrol, and thymol were present in the BCSO. In trial 1 with strain Cp#6, BCSO concentrations of 2 and 5 mL/kg reduced NE lesion score and mortality rate to 1.6% compared with 7.8% for positive control, with no adverse impact on live performance. In trial 2 with strain Cp#4, BCSO reduced NE lesion scores and mortality rate to 35.9% compared with 51.6% for positive control and also improved weight gain when there was a Cp infection in broiler chickens. The current study compared NE in broilers challenged with 2 different Cp strains producing different levels of NE. Following Cp infection, both the population of vegetative cells and spores of Cp in cecal contents decreased for all treatments in trial 2. In conclusion, BCSO at concentrations of 2 and 5 mL/kg enhanced broiler live performance and alleviated NE and has potential as a natural, non-medication antimicrobial nutritional supplement for use as a feed additive in chickens.

Biotransformation kinetics and pathways of typical synthetic progestins in soil microcosms.

Gestodene (GES), altrenogest (ALT), and medroxyprogesterone acetate (MPA) are three potent synthetic progestins detected in agricultural soils; however, their biotransformation outcomes in soils remain unclear. This study explored the biotransformation of these progestins in five agricultural soils with different physicochemical properties. The biotransformation data were well-described by a first-order decay model (R2 = 0.83-0.99), with estimated half-lives ranging between 12.1 and 188 h. Amplicon sequencing indicated that the presence of progestins changed the bacterial richness and community structure in the soils. Linear correlation, canonical correlation, and two-way correlation network analysis revealed that soil properties can affect biotransformation rates by interfering with progestin-soil interactions or with keystone taxa in soils. The clustermap demonstrated the formation of abundant transformation products (TPs). Isomerization and C4(5) hydrogenation were the major transformation pathways for GES (yields of ∼ 13.7 % and ∼ 10.6 %, respectively). Aromatic dehydrogenation was the major transformation pathway for ALT (yield of ∼ 17.4 %). The C17 hydrolysis with subsequent dehydration and hydrogenation was the major transformation pathway for MPA (yield of ∼ 196 %). In particular, some TPs exhibited progestagenic, androgenic, or estrogenic activity. This study highlights the importance of evaluating the ecotoxicity of progestin and TP mixtures for better understanding their risks in the environment.

Abiotic transformation of synthetic progestins in representative soil mineral suspensions.

Altrenogest (ALT), drospirenone (DRO), and melengestrol acetate (MLA) are three highly potent synthetic progestins that can be released into agricultural soils, while their fate in soil minerals remains unclear. This study explored the transformation of these progestins in MnO2, SiO2, and ferrihydrite suspensions and identified their transformation products (TPs) via high resolution mass spectrometry and density functional theory calculations. Transformations were only observed for DRO and MLA in SiO2 suspension and ALT in MnO2 suspension (half-lives = 0.86 min - 9.90 day). ALT transformation was facilitated at higher MnO2 loadings, while DRO and MLA transformations were inhibited at higher SiO2 loadings. These data indicated that hydrophobic partitioning interaction was dominant at higher SiO2 loadings rather than specific interaction, which limited subsequent surface-catalyzed transformation. ALT transformation rate decreased with increasing pH because MnO2 reduction requires proton participation. In contrast, relatively high pH facilitated MLA and DRO transformation, indicating that base-catalyzed hydrolysis occurred in SiO2 suspension. The clustermap demonstrated the formation of abundant TPs. Lactone ring and acetoxy group hydrolysis was the major transformation pathway for DRO and MLA, with estimated yields of 57.7% and 173.2% at 6 day, respectively. ALT experienced C12 hydroxylation and formed the major TP 326g (yield of 15.4% at 8 hr). ALT also experienced allyl group oxidation and subsequent C5 hydroxylation, forming the major TP 344a (yield of 14.1% at 8 hr). This study demonstrates that TPs of metastable progestins are likely the main species in soils and that TP identification is a particular priority for risk assessment.

The maternal hair metabolome is capable of discriminating intrahepatic cholestasis of pregnancy from uncomplicated pregnancy.

Introduction: Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disease associated with elevated bile acids in the blood. Diagnosis typically only occurs after the manifestation of clinical symptoms and the metabolic mechanisms underlying its development remain unclear. The aim of this study was to investigate potential specific metabolites and the underlying metabolic changes occurring during the development of ICP in the maternal plasma and hair metabolomes of women diagnosed with either ICP or having a healthy pregnancy. Methods: A total of 35 Chinese women with ICP and 42 healthy pregnancies were enrolled in our study. Plasma and hair samples, total bile acid levels (TBA), alanine transaminase levels (ALT), aspartate aminotransferase levels (AST), and additional clinical information were collected during the third trimester. Metabolites from maternal plasma and hair segments collected pre-conception and analyzed using gas chromatography-mass spectrometry (GC-MS). Results: Three plasma metabolites (p < 0.05, q < 0.38) and 21 hair metabolites (p < 0.05, q < 0.05) were significantly different between ICP and healthy pregnancies. A combination of the eight most significant hair metabolites in a multivariate receiver operating characteristic curve model showed the best area under the curve (AUC) was 0.885, whereas the highest AUC using metabolites from plasma samples was only 0.74. Metabolic pathway analysis revealed 32 pathways were significantly (p and q values < 0.05) affected in the hair samples of patients with ICP. Pathways associated with glutathione metabolism and ABC transporters were affected. No metabolic pathways were significantly affected in plasma. Discussion: Overall, this study showed that the hair metabolome could be more useful than the plasma metabolome for distinguishing ICP from normal pregnancy.

Robust and Transparent Silver Oxide Coating Fabricated at Room Temperature Kills Clostridioides difficile Spores, MRSA, and Pseudomonas aeruginosa.

Antimicrobial coatings can inhibit the transmission of infectious diseases when they provide a quick kill that is achieved long after the coating application. Here, we describe the fabrication and testing of a glass coating containing Ag2O microparticles that was prepared from sodium silicate at room temperature. The half-lives of both methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa on this coating are only 2-4 min. The half-life of Clostridioides difficile spores is about 9-12 min, which is extremely short for a spore. Additional tests on MRSA demonstrate that the coating retains its antimicrobial activity after abrasion and that an increased loading of Ag2O leads to a shorter half-life. This coating combines the properties of optical transparency, robustness, fast kill, and room temperature preparation that are highly desirable for an antimicrobial coating.

Compositional and Functional Changes in Microbial Communities of Composts Due to the Composting-Related Factors and the Presence of Listeria monocytogenes.

Listeria monocytogenes is a leading foodborne pathogen that can contaminate fresh produce in farm environment, resulting in deadly outbreaks. Composts contain a diversity of microorganisms, and some of them may be compost-adapted competitive exclusion microorganisms against L. monocytogenes. To understand interactions between compost microflora and the pathogen, both dairy- and poultry-wastes based composts (n = 12) were inoculated with L. monocytogenes, and then analyzed by next-generation sequencing approaches along with culturing methods. DNA extraction and enumeration of L. monocytogenes were performed at 0 and 72 h post-incubation at room temperature. The major bacterial phyla were identified as Firmicutes (23%), Proteobacteria (23%), Actinobacteria (19%), Chloroflexi (13%), Bacteroidetes (12%), Gemmatimonadetes (2%), and Acidobacteria (2%). The top three indicator genera enriched in different compost types were identified by LEfSe with LDA score > 2. The interactions between L. monocytogenes and indigenous microflora were limited as no significant changes in the dominant microbial members in compost ecosystem, but some discriminatory species such as Bacillus, Geobacillus, and Brevibacterium were identified by Random Forest analysis. Besides, changes in metabolic pathways and the increased abundance of bacteriocins category in the compost samples containing L. monocytogenes after 72 h postinoculation were revealed by metatranscriptomic sequencing. Taken together, the compost-related factors such as compost types, composting stages, and the collection farms are major drivers that affect compost microbial compositions, and the analysis of compost metagenome implied that interactions between L. monocytogenes and compost microflora may include competition for nutrients and the presence of antimicrobials. IMPORTANCE Listeria monocytogenes has been recognized as the etiological agent causing foodborne disease outbreaks, with fresh produce as vulnerable for contamination at even preharvest stage. Owing to the richness in microbial community, compost may mediate suppression of pathogens. In this study, the impact of compost-related factors and L. monocytogenes intrusion on dynamic changes in compost microbiome was investigated by next generation sequencing techniques. The compost-related factors such as compost types, composting stages, and the collection farms are major drivers that affect compost microbiome. The interactions between L. monocytogenes and compost microflora may include the competition for nutrients and the presence of antimicrobials produced by native compost microorganisms as potential competitive exclusion microorganisms. Findings from this study are important for the composting industry to understand the composition and functionality of microbial community in their products and help developing organic fertilizers fortified with anti-L. monocytogenes competitive exclusion microorganisms.

Efficacy of EPA-registered disinfectants against two human norovirus surrogates and Clostridioides difficile endospores.

AIMS: To determine the efficacy of a panel of nine EPA-registered disinfectants against two human norovirus (HuNoV) surrogates (feline calicivirus [FCV] and Tulane virus [TuV]) and Clostridioides difficile endospores. METHODS AND RESULTS: Nine EPA-registered products, five of which contained H2 O2 as active ingredient, were tested against infectious FCV, TuV and C. difficile endospores using two ASTM methods, a suspension and carrier test. Efficacy claims against FCV were confirmed for 8 of 9 products. The most efficacious product containing H2 O2 as ingredient achieved a >5.1 log reduction of FCV and >3.1 log reduction of TuV after 5 min, and >6.0 log reduction of C. difficile endospores after 10 min. Of the five products containing H2 O2 , no strong correlation (R2  = 0.25, p = 0.03) was observed between disinfection efficacy and H2 O2 concentration. Addition of 0.025% ferrous sulphate to 1% H2 O2 solution improved efficacy against FCV, TuV and C. difficile. CONCLUSION: Disinfectants containing H2 O2 are the most efficacious disinfection products against FCV, TuV and C. difficile endospores. Product formulation, rather than the concentration of H2 O2 in a product, impacts the efficacy of a disinfection product. SIGNIFICANCE AND IMPACT OF STUDY: H2 O2 -based disinfectants are efficacious against surrogate viruses for HuNoV and C. difficile endospores.

Isolation and characterization of competitive exclusion microorganisms from animal wastes-based composts against Listeria monocytogenes.

AIM: To isolate the slow-growing or viable but non-culturable competitive exclusion (CE) microorganisms from composts and then verify the anti-Listeria monocytogenes activities of those CE isolates in compost. METHODS AND RESULTS: CE strains were isolated from composts using double- or triple-layer agar methods, purified, and then characterized. Both compost extracts and solid compost samples were spiked with a cocktail of 3 L. monocytogenes strains which were co-inoculated with or without CE strain cocktail and incubated at both 22 and 35°C for 168 h. Results indicated that the addition of resuscitation promoting factor (Rpf) promoted the growth of slow-growing species from composts. About 50% of the isolated CE strains (n = 40) were identified as Bacillus spp., 17 strains can inhibit more than 10 tested L. monocytogenes strains, and nine strains were motile and competitive biofilm formers. In compost extracts, the growth potentials of L. monocytogenes were reduced up to 2.2 logs when co-culturing with CE strains. In compost samples, the addition of CE strains reduced L. monocytogenes population by ca. 1.3 log CFU/g at 22°C after 24-168 h incubation. CONCLUSION: Our modified double/triple-layer agar procedure with Rpf as growth supplement coupled with spot-on-lawn testing can be a quick and efficient method for isolating CE candidates from composts. The efficacy of CE strains against L. monocytogenes in compost extracts and compost samples was affected by compost type, nutrient level, and incubation temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: Compost is a rich source of CE microorganisms, and compost-adapted CE microorganisms have the potential as a biological agent to control L. monocytogenes in agricultural environments.

Plant-Scale Validation of Physical Heat Treatment of Poultry Litter Composts Using Surrogate and Indicator Microorganisms for Salmonella.

This study selected and used indicator and surrogate microorganisms for Salmonella to validate the processes for physically heat-treated poultry litter compost in litter processing plants. Initially laboratory validation studies indicated that 1.2- to 2.7-log or more reductions of desiccation-adapted Enterococcus faecium NRRL B-2354 were equivalent to > 5-log reductions of desiccation-adapted Salmonella Senftenberg 775/W in poultry litter compost, depending on treatment conditions and compost types. Plant validation studies were performed in one turkey litter compost processor and one laying hen litter compost processor. E. faecium was inoculated at ca.7 log CFU g-1 into the turkey litter compost and at ca. 5 log CFU g-1 into laying hen litter compost with respectively targeted moisture contents. The thermal processes in the two plants yielded 2.8 - > 6.4 log CFU g-1 (> 99.86%) reductions E. faecium of the inoculated. Similarly, for the processing control samples, reductions of presumptive indigenous enterococci were in the order of 1.8-3.7 log CFU g-1 (98.22% to 99.98%) of the total naturally present. In contrast, there were less reductions of indigenous mesophiles (1.7-2.9 log CFU) and thermophiles (0.4-3.2 log CFU g-1). More indigenous enterococci were inactivated in the presence of higher moisture in the poultry litter compost. Based on the data collected under the laboratory conditions, the processing conditions in both plants were adequate to reduce any potential Salmonella contamination of processed poultry litter compost by at least 5 logs, even though the processing conditions varied among trials and plants.IMPORTANCE Poultry litter compost, commonly used as a biological soil amendment, is subjected to a physical heat-treatment in industry setting to reduce pathogenic bacteria such as Salmonella and produce a dry product. According to the FDA Food Safety Modernization Act (FSMA) Produce Safety Rule, the thermal process for poultry litter compost should be scientifically validated to satisfy the microbial standard requirement. To the best of our knowledge, this is the first validation study in commercial poultry litter compost processing plants, and our results indicated that Salmonella levels, if present, could be reduced by at least 5 logs based on the reductions of surrogate and indicator microorganisms, even though the processing conditions in these commercial plants varied greatly. Furthermore, both indicator and surrogate microorganisms along with the custom-designed sampler can serve as practical tools for poultry litter compost processors to routinely monitor or validate their thermal processes without introducing pathogens into the industrial environments.

Sorption, transport, and transformation of natural and synthetic progestins in soil-water systems.

Natural and synthetic progestins are emerging endocrine disruptors that can be transported from livestock farms and agricultural fields to receiving waters via surface runoff. The transformation of progestins during transport is expected to affect the efficiencies of runoff management systems. Therefore, this study explored the sorption, transport, and transformation of progesterone, norethisterone acetate, medroxyprogesterone acetate, cyproterone acetate, dydrogesterone, and norethisterone in agricultural soil-water systems. The sorption coefficients and retardation factors (R) were positively correlated with the progestin hydrophobicities, indicating that hydrophobic interactions dominated the sorption and transport processes. During transport, dydrogesterone and progesterone were transformed into 9-10 products. The breakthrough curves of the parents and products exhibited periodical patterns over extended times. Specifically, the R values of the parents and products were positively correlated with chromatographic retention times (hydrophobicities) when the products were generated before transport. In contrast, a negative correlation (R2 = 0.75-0.88) was observed when products were successively generated during transport, indicating that the transformation kinetics changed the retardation of these solutes in the columns. These observations also demonstrated that the transport potential estimates based on traditional metrics of steroid hydrophobicity are not always accurate and that runoff management measures are less effective for metastable progestins.

Composting To Inactivate Foodborne Pathogens for Crop Soil Application: A Review.

Compost is organic material that has been degraded into a nutrient-stabilized humus-like substance through intense microbial activity, which can provide essential plant nutrients (nitrogen, phosphorus) to aid in the growth of fruits and vegetables. Compost can be generated from animal waste feedstocks; these can contain human pathogens, which can be inactivated through the heat and microbial competition promoted during the composting process. Outbreaks of infections caused by bacterial pathogens such as Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes on fruit and vegetable commodities consumed raw emphasize the importance of minimizing the risk of pathogenic contamination on produce commodities. This review article investigates factors that affect the reduction and survival of bacterial foodborne pathogens during the composting process. Interactions with indigenous microorganisms, carbon:nitrogen ratios, and temperature changes influence pathogen survival, growth, and persistence in finished compost. Understanding the mechanisms of pathogen survival during the composting process and mechanisms that reduce pathogen populations can minimize the risk of pathogen contamination in the cultivation of fruits and vegetables.

Testing a Nonpathogenic Surrogate Microorganism for Validating Desiccation-Adapted Salmonella Inactivation in Physically Heat-Treated Broiler Litter.

Thermal resistance of desiccation-adapted Salmonella Senftenberg 775W was compared with that of Enterococcus faecium NRRL B-2354 in aged broiler litter. Aged broiler litter with 20, 30, and 40% moisture contents was inoculated separately with desiccation-adapted Salmonella Senftenberg 775W and E. faecium NRRL B-2354 at ca. 5 to 6 log CFU/g and then heat treated at 75, 85, and 150°C. At all tested temperatures, desiccation-adapted E. faecium NRRL B-2354 was more heat resistant than desiccation-adapted Salmonella Senftenberg 775W ( P < 0.05). During the treatments at 75 and 85°C, E. faecium NRRL B-2354 in aged broiler litter with all moisture contents was reduced by 2.89 to 4.12 log and was above the detection limit of direct plating (1.30 log CFU/g), whereas Salmonella Senftenberg 775W could not be detected by enrichment (>5-log reduction) during holding time at these temperatures. At 150°C, E. faecium NRRL B-2354 in aged broiler litter with 20 and 30% moisture contents was still detectable by enrichment after heat exposure for up to 15 min, whereas Salmonella Senftenberg 775W in aged broiler litter with all moisture contents could not be detected throughout the entire treatment. Our results revealed that E. faecium NRRL B-2354 can be used as a surrogate for Salmonella to validate the thermal processing of poultry litter by providing a sufficient safety margin. This study provides a practical tool for poultry litter processors to evaluate the effectiveness of their thermal processing.

Complete Genome of a Novel Lytic Vibrio parahaemolyticus Phage VPp1 and Characterization of Its Endolysin for Antibacterial Activities.

Vibrio parahaemolyticus is an important foodborne pathogen that is generally transmitted via raw or undercooked seafood. Endolysins originating from bacteriophages offer a new way to control bacterial pathogens. The objectives of this study were to sequence a novel lytic V. parahaemolyticus phage VPp1 and determine the antibacterial activities of the recombinant endolysin (LysVPp1) derived from this phage. The complete VPp1 genome contained a double-stranded DNA of 50,431 bp with a total G+C content of 41.35%. The genome was predicted to encode 67 open reading frames (ORFs), which were organized as nucleotide metabolism, replication, structure, packaging, lysis, and some additional functions. Two tRNAs were encoded to carry anticodons UGG and CCA. Among the functional proteins, ORF33 was deduced to encode endolysin, whereas no holin/antiholin or Rz/Rz1 lysis gene equivalents were found in the VPp1 genome. ORF33 was cloned and expressed. The endolysin LysVPp1 could lyse 9 of 12 V. parahaemolyticus strains, showing its relatively broader host spectrum than phage VPp1, which lysed only 3 of 12 V. parahaemolyticus strains. Furthermore, for EDTA-pretreated bacterial cells, the optical density of the LysVPp1 treatment group decreased by 0.4 at 450 nm, compared with less than 0.1 in control groups, demonstrating enhanced hydrolytic properties. These results contribute to the potential for development of novel enzybiotics for controlling V. parahaemolyticus.

Isolation of Toxigenic Clostridium difficile from Animal Manure and Composts Being Used as Biological Soil Amendments.

The well-known nosocomial pathogen Clostridium difficile has recently been recognized as a community-associated pathogen. As livestock animals carry and shed C. difficile in their feces, animal manure-based composts may play an important role in disseminating toxigenic C. difficile strains into the agricultural environment. The present study surveyed C. difficile contamination of commercially available composts and animal manure. Presumptive C. difficile isolates were confirmed by testing for the tpi housekeeping gene in addition to Gram staining. The confirmed C. difficile isolates were further tested for toxigenicity, PCR ribotype, and susceptibilities to selected antibiotics. C. difficile was found in 51 out of 142 samples (36%). A total of 58 C. difficile strains were isolated from those 51 positive compost/manure samples. The presence of C. difficile in compost did not significantly correlate (P > 0.05) with the physical and most microbiological parameters, including the presence of fecal coliforms. The majority of C. difficile isolates were toxigenic, with 63.8% positive for the toxin A gene (tcdA) and 67.2% positive for the toxin B gene (tcdB). Only 3 isolates (5.17%) were positive for the binary toxins. There were 38 different PCR ribotypes among the 58 C. difficile isolates, and ribotype 106 was the most prevalent, followed by ribotypes 020 and 412. All C. difficile isolates were susceptible to the selected antibiotics, but >50% of the isolates had reduced susceptibility to clindamycin by the agar dilution method. This study indicates that compost may be a reservoir of toxigenic C. difficile strains.IMPORTANCEClostridium difficile infection (CDI) is a leading cause of antibiotic-associated diarrhea in health care facilities in developed countries. Extended hospital stays and recurrences severely increase the cost of treatments and the high mortality rate that is observed among the elderly. Community-associated CDI cases that occur without any recent contact with the hospital environment are increasing. Studies have reported the isolation of virulent C. difficile strains from water, soil, meat, vegetables, pets, livestock animals, and animal manure. The objective of this study was to isolate and characterize C. difficile strains from animal manure and commercially available compost products. Our results demonstrate that not only unprocessed animal manure but also finished composts made of different feedstocks can serve as a reservoir for C. difficile as well. Most importantly, our study revealed that properly processed compost is a potential source of toxigenic and clindamycin-resistant C. difficile isolates.

Comparative Recovery of Two Human Norovirus Surrogates, Feline Calicivirus and Murine Norovirus, with a Wet Vacuum System, Macrofoam-Tipped Swab, and Bottle Extraction Method from Carpets.

Human noroviruses (HuNoV) are the leading cause of known foodborne illness in the United States, but direct detection during outbreak investigations is challenging. On the other hand, sampling both hard and soft environmental surfaces can be used to improve outbreak investigations. Currently, we lack virus recovery methods for soft surfaces, such as carpet, despite evidence suggesting that carpets contribute to HuNoV outbreaks. Our aim was to compare two recovery methods, wet vacuum and swabbing, for routine carpet sampling of HuNoV against a laboratory reference method known as bottle extraction (BE). Specifically, we compared the microbial vacuum (MVAC), macrofoam-tipped swab (MS), and BE by using HuNoV surrogates, feline calicivirus (FCV) and murine norovirus (MNV), inoculated on wool and nylon carpet carriers. The highest recovery of infectious FCV from wool was 5.51, 3.76, and 5.16 log PFU, whereas on nylon, recovery was 5.03, 3.62, and 4.75 log PFU by using BE, MS, and MVAC, respectively. On the other hand, the highest recovery of infectious MNV from wool was 6.10, 4.50, and 5.99 log PFU, while recovery on nylon was 6.07, 4.58, and 6.13 log PFU by using BE, MS, and MVAC, respectively. Significantly more PFU and genomic copies were recovered by using BE and MVAC compared with MS, while buffer type played a significant role in recovery of infectious FCV.