Spore-forming bacteria in gelatin: Characterization, identification by 16S rRNA and MALDI-TOF mass spectrometry (MS), and presence of heat resistance and virulence genes.

Gelatin, a versatile protein derived from collagen, is widely used in the food, pharmaceutical and medical sectors. However, bacterial contamination by spore-forming bacteria during gelatin processing represents a significant concern for product safety and quality. In this study, an investigation was carried out to explore the heat and chemical resistance, as well as the identification and characterization of spore-forming bacteria isolated from gelatin processing. The methodologies involved chemical resistance tests with drastic pH in microplates and thermal resistance tests in capillary tubes of various isolates obtained at different processing stages. In addition, phenotypic and genotypic analyses were carried out to characterize the most resistant isolates of spore-forming bacteria. The findings of this study revealed the presence of several species, including Bacillus cereus, Bacillus licheniformis, Bacillus sonorensis, Bacillus subtilis, Geobacillus stearothermophilus, and Clostridium sporogenes, with some isolates exhibiting remarkable chemical and heat resistances. In addition, a significant proportion of the most resistant isolates showed gelatinase activity (n = 19/21; 90.5 %) and the presence of heat resistance (n = 5/21; 23.8 %), and virulence genes (n = 11/21; 52.4 %). The results of this study suggest that interventions should be done in quality control practices and that process parameter adjustments and effective contamination reduction strategies should be implemented through gelatin processing.

Solar/ClO2 system inactivates fungal spores in drinking water: Synergy, efficiency and mechanisms.

The risk of fungal pollution in drinking water has been paid attention. Solar/chlorine dioxide (ClO2) combined system is an environment-friendly, economical and efficient disinfection method, especially for countries and regions that are economically backward and still exposed to unsafe drinking water. In this paper, the kinetics, influencing factors, mechanism and regrowth potential of inactivated Aspergillus niger (A. niger) spores by solar/ClO2 were reported for the first time. The inactivation curve can be divided into three stages: instant inactivation within 1-2 min, slow linear inactivation and finally a tail. The synergistic factors produced by solar/ClO2 in terms of log reduction and maximum inactivation rate were 1.194 and 1.112, respectively. The inhibitory effect on the regrowth of A. niger spores inactivated by solar/ClO2 was also stronger than that by ClO2 alone. Strongly oxidizing reactive species produced by solar/ClO2 accelerated the accumulation of endogenic reactive oxygen species (ROS) caused by oxidation stress of A. niger spores, improving the inactivation ability of the system. The inactivation order of A. niger spores was: loss of culturability, accumulation of intracellular ROS, loss of membrane integrity, leakage of intracellular species and change of morphology. The inactivation performance of solar/ClO2 was better than solar/chlor(am)ine according to the comparison of inactivation efficiency and regrowth potential. Results also suggested that solar/ClO2 process was more suitable for the treatment of ground water sources.

[Identification and sensitization to environmental fungal spores in Lima City, Peru]. / Identificación y sensibilización a hongos ambientales en Lima, Perú.

OBJECTIVE: To identify and registry the most important fungal spores trapped in our aerobiology station, as well as to report the prevalence of skin sensitization to these allergens. METHODS: The pollen counts were made according to standardized technique with a Burkard seven days spore trap, following the American Academy of Allergy, Asthma and Immunology (AAAAI) through National Allergy Bureau (NAB) recommendations. The trap was installed on the roof of Clinica SANNA, El GOLF, San Isidro, which is 20 m high, 12°5'54"S 77°3'6"W in the west-south of the Lima urban area. The sampling period was performed from September 2020 to October 2021. Skin prick tests were carried out according to the recommendations of the Spanish Society of Allergology and Clinical Immunology (SEAIC) in 200 patients (18 to 60 years old) with symptoms of rhinoconjunctivitis and/or asthma, who were evaluated in the Allergology Service of Clinica SANNA el Golf. Allergenic extracts were applied, dust mites (Dermatophagoides pteronyssinus, Dermatophagoides farinae, Blomia tropicalis), cat and dog danders, cockroach (Periplaneta americana), grass 6 mix, weed mix, molds (Cladosporium herbarum, Alternaria alternata, Aspergillus fumigatus, Penicillium notatum, Nigrospora spp.), INMUNOTEK-Spain provided the extracts. We also tested other fungal allergens such as Fusarium spp, Stemphylium spp, Curvularia spp, a mixture of Helmintosporum/Dreschlera spp. from the DIATER-Argentina laboratory. RESULTS: We identified spores of Alternaria alternata, Cladosporium spp., Nigrospora spp., Stemphylium spp., Fusarium spp., Curvularia spp., Dreschlera/Helmintosporum spp. The patients showed sensitization to Cladosporium herbarum (14%), Fusarium spp. (13,5%), Nigrospora spp. (8%), Alternaria Alternata (7%), Stemphylium (6%), Dreschlera/Helmintosporium spp. (5,5%), Curvularia spp. (3%), Aspergillus fumigatus (2,5%). CONCLUSIONS: The inhabitants of the south-western area of the urban city of Lima are exposed to different fungal spores with allergenic potential, with a higher concentration being identified during the summer/autumn months. Cutaneous sensitization is demonstrated in variable percentages to the fungal spores identified in this aerobiological sampling. The results of this study should be expanded and compared with data in the forthcoming years, identify seasonal and annual fluctuations and extend the traps to other locations in Lima.

OBJETIVO: Identificar y registrar las esporas de hongos más importantes captadas en nuestra estación de aerobiología, además reportar la prevalencia de sensibilización cutánea a estos alérgenos. MÉTODOS: La identificación y los conteos de esporas de hongos se realizaron según la técnica estandarizada con un equipo colector Burkard Spore Trap For Seven Days, siguiendo las recomendaciones de la National Allergy Bureau (NAB), de la American Academy Allergy Asthma and Immunology (AAAAI). El equipo se instaló a 20 m de altura desde el nivel del suelo, en la azotea de la Clínica SANNA El Golf, distrito de San Isidro, (12°5'54"S 77°3'6"O), en la zona sur-oeste del área urbana de Lima. El periodo de captación se llevó a cabo entre septiembre de 2020 y octubre de 2021. Se realizaron estudios de pruebas cutáneas (skin prick-test), según recomendaciones de la Sociedad Española de Alergología e Inmunología Clínica (SEAIC), en 200 pacientes (entre 18 y 60 años), con sintomatología de rinoconjuntivitis y/o asma. Fueron evaluados en el servicio de Alergología de la Clínica SANNA El Golf. Se aplicaron extractos alergénicos de ácaros del polvo (Dermatophagoides pteronyssinus, Dermatophagoides farinae, Blomia tropicalis), epitelios de gato y perro, Periplaneta americana, mezclas de seis gramíneas, mezclas de malezas, hongos ambientales (Cladosporium herbarum, Alternaria alternata, Aspergillus fumigatus, Penicillium notatum, Nigrospora spp.), extractos del laboratorio INMUNOTEK-España. Además, testeamos otros alérgenos fúngicos de Fusarium spp, Stemphylium spp, Curvularia spp, una mezcla de Helmintosporum/Dreschlera spp. del laboratorio DIATER-Argentina. RESULTADOS: Identificamos esporas de Alternaria alternata, Cladosporium spp., Nigrospora spp., Stemphylium spp., Fusarium spp., Curvularia spp., Dreschlera/Helmintosporum spp. Los pacientes mostraron sensibilización a Cladosporium herbarum (14%), Fusarium spp. (13,5%), Nigrospora spp. (8%), Alternaria Alternata (7%), Stemphylium (6%), Dreschlera/Helmintosporium spp. (5,5%), Curvularia spp. (3%) y Aspergillus fumigatus (2,5%). CONCLUSIONES: Los habitantes de la zona sur-oeste de la ciudad urbana de Lima están expuestos a distintas esporas de hongos con potencial alergénico, identificándose mayor concentración durante los meses de verano y otoño. Se demuestra sensibilización cutánea en porcentajes variables a las esporas fúngicas identificadas en este muestreo aerobiológico. Los resultados de este estudio deberían ampliarse y ser comparados con data en los años siguientes, identificar fluctuaciones estacionales y anuales y extender los captadores a otras locaciones en Lima.

Effectiveness of Bacillus subtilis ANT01 and Rhizobium sp. 11B on the control of fusarium wilt in pineapple (Ananas comosus).

Pineapple (Ananas comosus) is commonly infected by Fusarium oxysporum, causal agent of the fusarium wilt disease. Conventionally, growers use synthetic fungicides to control the disease, which lead to environmental pollution, hazardous effects on non-target organisms and risks on human health. The aim of this work was to assess the effectiveness of Bacillus subtilis ANT01 and Rhizobium sp. 11B to control fusarium wilt on pineapple plants. Four treatments derived from a complete factorial design were tested under field conditions. Treatments composed of B. subtilis ANT01 and the combination B. subtilis ANT01-Rhizobium sp. 11B decreased disease severity by 94.4% and 86.1%, respectively. On the other hand, the treatment prepared with Rhizobium sp. 11B alone showed a reduction of 75.0%. Size of leaves and nutritional condition (SPAD units) of the biocontrol agents-treated plants showed no statistical differences. Moreover, B. subtilis ANT01 decreased by 46% the initial soil population of F. oxysporum, while Rhizobium sp. 11B, B. subtilis ANT01 plus Rhizobium sp. 11B and control, showed a population reduction of 12.5%, 24.2% and 23.0%, respectively. These results make evident the potential of B. subtilis ANT01 as biocontrol agent of the pathogen under field conditions.

Counts of mesophilic aerobic, mesophilic anaerobic, thermophilic aerobic sporeforming bacteria and persistence of Bacillus cereus spores throughout cocoa powder processing chain.

Sporeforming bacteria are a concern in some food raw materials, such as cocoa powder. Samples (n = 618) were collected on two farms and at several stages during cocoa powder manufacture in three commercial processing lines to determine the impact of each stage on bacterial spore populations. Mesophilic aerobic, mesophilic anaerobic, thermophilic aerobic, and Bacillus cereus spore populations were enumerated in all the samples. Genetic diversity in B. cereus strains (n = 110) isolated from the samples was examined by M13 sequence-based PCR typing, partial sequencing of the panC gene, and the presence/absence of ces and cspA genes. The counts of different groups of sporeforming bacteria varied amongst farms and processing lines. For example, the counts of mesophilic aerobic spore-forming (MAS) populations of cocoa bean fermentation were lower than 1 log spore/g in Farm 1 but higher than 4 log spore/g in Farm 2. B. cereus isolated from cocoa powder was also recovered from cocoa beans, nibs, and samples after roasting, refining, and pressing, which indicated that B. cereus spores persist throughout cocoa processing. Phylogenetic group IV was the most frequent (73%), along with processing. Strains from phylogenetic group III (14 %) did not show the ces gene's presence.

Beauveria bassiana submerged spores for control of two-spotted spider mite (Tetranychus urticae Koch (Acari: Tetranychidae)): production, stability, and virulence.

In this study, IBCB 66, IBCB 868, and CBMAI 1306 isolates of Beauveria bassiana were grown in liquid culture for 4 days, leading to elevated submerged spores (SS) levels. The influence of the addition of different glycerol concentrations (0, 3, and 6%) (v/v) in the liquid culture was investigated regarding the stability (at 4 and 27 °C) of dried formulations. The virulence of SS was compared with aerial spores (AS) against Tetranychus urticae (Koch) (Acari: Tetranychidae). The results demonstrate the potential of using SS to control T. urticae. CBMAI 1306 and IBCB 868 isolates caused T. urticae mortality rates of 91.11% and 88.89% 5 days after treatment, respectively, when applied at concentrations of 1 × 108 SS mL-1. The median Lethal Time (LT50) values for these strains were 2.64 and 2.61 days, respectively. The dried formulations showed potential acaricidal activity. Higher glycerol concentrations in the liquid culture medium reduced formulation stability at 27 °C.

Fungal spores in Caribbean mangrove sediments, dataset from southeastern Mexico.

Most paleoecological investigations use different biotic or abiotic proxies for climate and environmental reconstructions. Although fossil pollen is one of the most used biological proxies, Non-Pollen Palynomorphs (NPPs), especially fungal spores and tissues, have an underestimated potential to infer local and regional climate dynamics. This dataset describes the most common Non-pollen palynomorphs of fungal origin from mangrove sediments in the Caribbean Sea, southeastern Mexico. A detailed descriptive Atlas is presented, with light micrographs taken from routine pollen slides in paleoecological reconstructions. Microphotographs were included to facilitate their identification. A total of 59 spores, 4 tissues, 2 hyphae, and 11 unidentified fungal palynomorphs are described.

The chronicle of the ups and downs that made Bacillus thuringiensis a natural insecticide

Bacillus thuringiensis is a worldwide known bacterium for its capacity to control insect pests thanks to the action of its parasporal crystal. The objective of this paper deals with the history, in some cases unknown, of the study of Bacillus thuringiensis that led it to be a crucial biological alternative in controlling pest insects. How the mode of action for killing insects was understood, as well as the field tests that were carried out to evaluate its effectiveness and to develop the first commercial products, are reflected in this review that presents and discusses the scientific successes and failures that marked the course of B. thuringiensis.

Bacillus thuringiensis es una bacteria conocida mundialmente por su capacidad para controlar insectos plaga, gracias a la acción de su cristal parasporal. El objetivo de esta revisión trata de la historia, en algunos casos desconocida, del estudio de Bacillus thuringiensis que la llevó a ser una importante alternativa biológica en el control de insectos plaga. Cómo se llegó a comprender el modo de acción para matar insectos, así como las pruebas de campo que se realizaron para evaluar su efectividad y lograr desarrollar los primeros productos comerciales están plasmados en esta revisión que presenta y discute los aciertos y desaciertos científicos que marcaron el rumbo de B. thuringiensis.

Safety assessment of surfactin-producing Bacillus strains and their lipopeptides extracts in vitro and in vivo.

Beneficial Bacillus strains can be administered to livestock as probiotics to improve animal health. Cyclic lipopeptides produced by Bacillus such as surfactins may be responsible for some of the beneficial effects due to their anti-inflammatory and immunomodulatory activity. The aim of the present study was to isolate and evaluate the biocompatibility of native Bacillus spp. strains and their surfactin-like lipopeptides in vitro and in vivo to determine their potential to be used on animals. Biocompatibility of endospore suspensions (108 UFC/mL), and different dilutions (1:10; 1:50; 1:100; 1:500, and 1:1000) of Bacillus lipopeptide extracts containing surfactin was tested on Caco-2 cells by microculture tetrazolium-based colorimetric assay. Genotoxicity was tested on BALB/c mice (n = 6) administered 0.2 mL of endospore suspensions by the bone marrow erythrocyte micronuclei assay. All the isolates tested produced between 26.96 and 239.97 µg mL- 1 of surfactin. The lipopeptide extract (LPE) from isolate MFF1.11 demonstrated significant cytotoxicity in vitro. In contrast, LPE from MFF 2.2; MFF 2.7, TL1.11, TL 2.5, and TC12 had no cytotoxic effect (V% > 70%) on Caco-2 cells, not affecting cell viability signifficantly in most treatments. Similarly, none of the endospore suspensions affected cell viability (V% > 80%). Likewise, endospores did not cause genotoxicity on BALB/c mice. This study was elementary as a first step for a new line of research, since it allowed us to choose the safest isolates to keep working on the search of new potentially probiotic strains destined to production animals to improve their performance and health.

Assessment of fungal spores and spore-like diversity in environmental samples by targeted lysis.

At particular stages during their life cycles, fungi use multiple strategies to form specialized structures to survive unfavorable environmental conditions. These strategies encompass sporulation, as well as cell-wall melanization, multicellular tissue formation or even dimorphism. The resulting structures are not only used to disperse to other environments, but also to survive long periods of time awaiting favorable growth conditions. As a result, these specialized fungal structures are part of the microbial seed bank, which is known to influence the microbial community composition and contribute to the maintenance of diversity. Despite the importance of the microbial seed bank in the environment, methods to study the diversity of fungal structures with improved resistance only target spores dispersing in the air, omitting the high diversity of these structures in terms of morphology and environmental distribution. In this study, we applied a separation method based on cell lysis to enrich lysis-resistant fungal structures (for instance, spores, sclerotia, melanized yeast) to obtain a proxy of the composition of the fungal seed bank. This approach was first evaluated in-vitro in selected species. The results obtained showed that DNA from fungal spores and from yeast was only obtained after the application of the enrichment method, while mycelium was always lysed. After validation, we compared the diversity of the total and lysis-resistant fractions in the polyextreme environment of the Salar de Huasco, a high-altitude athalassohaline wetland in the Chilean Altiplano. Environmental samples were collected from the salt flat and from microbial mats in small surrounding ponds. Both the lake sediments and microbial mats were dominated by Ascomycota and Basidiomycota, however, the diversity and composition of each environment differed at lower taxonomic ranks. Members of the phylum Chytridiomycota were enriched in the lysis-resistant fraction, while members of the phylum Rozellomycota were never detected in this fraction. Moreover, we show that the community composition of the lysis-resistant fraction reflects the diversity of life cycles and survival strategies developed by fungi in the environment. To the best of our knowledge this is the first time that the fungal diversity is explored in the Salar de Huasco. In addition, the method presented here provides a simple and culture independent approach to assess the diversity of fungal lysis-resistant cells in the environment.

Inactivation of Alicyclobacillus acidoterrestris spores, single or composite Escherichia coli and native microbiota in isotonic fruit-flavoured sports drinks processed by UV-C light.

Pasteurized sports drinks and other fruit-based beverages are susceptible to deterioration due to thermal processing ineffectiveness to inactivate certain spoilage microorganisms, like Alicyclobacillus acidoterrestris. This represents a major challenge for the beverage industry. The goals of this study were to: i) investigate the UV-C inactivation (annular thin film unit, actinometrical delivered fluence: 795-1270 mJ/cm2, 10-15 min, 20 °C, 1.8 L/h, Reh = 391-1067, recirculation mode operation) and the evolution during refrigerated storage of A. acidoterrestris ATCC 49025 spores and single or composite Escherichia coli ATCC 25922 in isotonic sports drinks (ISDs) made from orange (orange-ISD, UVT% = 81) or orange-banana-mango-kiwi-strawberry-lemon juices (multi-fruit-ISD, UVT% = 91), compared to a turbid orange-tangerine juice (OT juice, UVT% = 40); ii) assess the effect of pH, °Brix, A254nm, turbidity, colour and particle size of the ISDs and juice on microbial inactivation, iii) evaluate the evolution of native microbiota during cold storage, iv) investigate the Coroller, biphasic, Weibull, and Weibull-plus-tail models' ability to describe microbial inactivation and v) measure 5-hydroxymethylfurfural (HMF) formation. The modified biodosimetry method was used to calculate the germicidal UV-C fluences. Heat pasteurization (T-coil, 80 °C/6 min) was evaluated as the control treatment. UV-C was highly effective at inactivating E. coli as 4.1-5.1 and 4.5-5.6 log reductions were determined in the multi-fruit-ISD and orange-ISD, respectively, barely impacted by the background microbiota. No significant differences were recorded for the inactivation of E. coli in the UV-C and T-coil systems. Whereas, a significantly higher inactivation of A. acidoterrestris spores was achieved by UV-C (3.7-4.0 log reductions), compared to the negligible one achieved by the thermal treatment. Even though E. coli inactivation curves were similar in shape, UV-C was less effective when a cocktail of other E. coli strains was present. In comparison to the OT juice, the ISDs' inactivation kinetics were markedly different in shape, with a rapid decrease in population during the first minutes of treatment. The germicidal fluence (Hd biod) corresponding to A. acidoterrestris (19.1 mJ/cm2) was selected as it was higher than the one obtained for E. coli (11.0 mJ/cm2). UV-C induced 2.8- or 1.3 and 2.3- or 0.8 log-reductions of total aerobes or moulds and yeasts in the multi-fruit-ISD and orange-ISD, respectively. Compared to the other models, the Coroller and biphasic models showed a better fit and more accurate parameter estimates. UV-C-induced HMF production was not significant in the ISDs. The current study found that the UV-C treatment was more effective than typical heat pasteurization for inactivating A. acidoterrestris spores in isotonic drinks, following a similar trend for E. coli and native microbiota.

Redistribution of the Novel Clostridioides difficile Spore Adherence Receptor E-Cadherin by TcdA and TcdB Increases Spore Binding to Adherens Junctions.

Clostridioides difficile causes antibiotic-associated diseases in humans, ranging from mild diarrhea to severe pseudomembranous colitis and death. A major clinical challenge is the prevention of disease recurrence, which affects nearly ~20 to 30% of the patients with a primary C. difficile infection (CDI). During CDI, C. difficile forms metabolically dormant spores that are essential for recurrence of CDI (R-CDI). In prior studies, we have shown that C. difficile spores interact with intestinal epithelial cells (IECs), which contribute to R-CDI. However, this interaction remains poorly understood. Here, we provide evidence that C. difficile spores interact with E-cadherin, contributing to spore adherence and internalization into IECs. C. difficile toxins TcdA and TcdB lead to adherens junctions opening and increase spore adherence to IECs. Confocal micrographs demonstrate that C. difficile spores associate with accessible E-cadherin; spore-E-cadherin association increases upon TcdA and TcdB intoxication. The presence of anti-E-cadherin antibodies decreased spore adherence and entry into IECs. By enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and immunogold labeling, we observed that E-cadherin binds to C. difficile spores, specifically to the hairlike projections of the spore, reducing spore adherence to IECs. Overall, these results expand our knowledge of how C. difficile spores bind to IECs by providing evidence that E-cadherin acts as a spore adherence receptor to IECs and by revealing how toxin-mediated damage affects spore interactions with IECs.

Role of the Spore Coat Proteins CotA and CotB, and the Spore Surface Protein CDIF630_02480, on the Surface Distribution of Exosporium Proteins in Clostridioides difficile 630 Spores.

Clostridioides difficile is Gram-positive spore-former bacterium and the leading cause of nosocomial antibiotic-associated diarrhea. During disease, C. difficile forms metabolically dormant spores that persist in the host and contribute to recurrence of the disease. The outermost surface of C. difficile spores, termed the exosporium, plays an essential role in interactions with host surfaces and the immune system. The main exosporium proteins identified to date include three orthologues of the BclA family of collagen-like proteins, and three cysteine-rich proteins. However, how the underlying spore coat influences exosporium assembly remains unclear. In this work, we explore the contribution of spore coat proteins cotA and cotB, and the spore surface protein, CDIF630_02480, to the exosporium ultrastructure, formation of the polar appendage and the surface accessibility of exosporium proteins. Transmission electron micrographs of spores of insertional inactivation mutants demonstrate that while cotB contributes to the formation of thick-exosporium spores, cotA and CDIF630_02480 contribute to maintain proper thickness of the spore coat and exosporium layers, respectively. The effect of the absence of cotA, cotB and CDIF630_02480 on the surface accessibility of the exosporium proteins CdeA, CdeC, CdeM, BclA2 and BclA3 to antibodies was affected by the presence of the spore appendage, suggesting that different mechanisms of assembly of the exosporium layer might be implicated in each spore phenotype. Collectively, this work contributes to our understanding of the associations between spore coat and exosporium proteins, and how these associations affect the assembly of the spore outer layers. These results have implications for the development of anti-infecting agents targeting C. difficile spores.

Environmental response of arbuscular mycorrhizal fungi under soybean cultivation at a regional scale.

Climate change, the shortage of fertilizers and reduced land for cultivation have drawn attention to the potential aid provided by soil-borne organisms. Arbuscular mycorrhizal fungi (AMF) offer a wide range of ecosystem benefits and hence, understanding the mechanisms that control AMF occurrence and maintenance is essential for resilient crop production. We conducted a survey of 123 soybean fields located across a 75,000-km2 area of Argentina to explore AMF community composition and to quantify the impact of soil, climate, and geographical distance on these key soil organisms. First, based upon morphological identification of spores, we compiled a list of the AMF species found in the studied area and identified Acaulospora scrobiculata and Glomus fuegianum as the most frequent species. G. fuegianum abundance was negatively correlated with precipitation seasonality and positively correlated with mean annual precipitation as well as mycorrhizal colonisation of soybean roots. Second, we observed that species richness was negatively correlated with soil P availability (Bray I), clay content and mean annual precipitation. Finally, based on partitioning variation analysis, we found that AMF exhibited spatial patterning at a broad scale. Therefore, we infer that geographical distance was positively associated with spore community composition heterogeneity across the region. Nevertheless, we highlight the importance of precipitation sensitivity of frequent species, overall AMF richness and community composition, revealing a crucial challenge to forthcoming agriculture considering an expected change in global climate patterns.

Biobased Spore Microcapsules for Asphalt Self-Healing.

Asphalt pavements and bituminous composites are majorly damaged by bitumen aging and fatigue cracking by traffic load. To add, maintenance and reparation of asphalt pavements is expensive and also releases significant amounts of greenhouse gases. These issues can be mitigated by promoting asphalt self-healing mechanisms with encapsulated rejuvenators. The ability of the required microcapsules to be resilient against high temperatures, oxidation, and mechanical stress is essential to promote such self-healing behavior without compromising the field performance of the asphalt pavement. This work proposes, for the first time, the use of extremely resistant biobased spores for the encapsulation of recycled oil-based rejuvenators to produce more resilient self-healing pavements. Spore encapsulants were obtained from natural spores (Lycopodium clavatum) by applying different chemical treatments, which enabled the selection of the best morphologically intact and clean spore encapsulant. The physical, morphological, and physicochemical changes were examined using fluorescence images, ATR-FTIR, SEM, size distribution, XRD, TGA and DSC analyses. Sunflower oil was used as the encapsulated rejuvenator with an optimal sol colloidal mixture for sporopollenin-oil of 1:5 (gram-to-gram). Vacuum, passive, and centrifugal encapsulation techniques were tested for loading the rejuvenator inside the clean spores and for selecting the best encapsulation technology. The encapsulation efficiency and the profiles of the accelerated release of the rejuvenator from the loaded spores over time were studied, and these processes were visualized with optical and inverted fluorescence microscopy. Vacuum encapsulation was identified as the best loading technique with an encapsulation efficiency of 93.02 ± 3.71%. The rejuvenator was successfully encapsulated into the clean spores, as observed by optical and SEM morphologies. In agreement with the TGA and DSC, the microcapsules were stable up to 204 °C. Finally, a self-healing test was conducted through fluorescence tests to demonstrate how these biobased spore microcapsules completely heal a crack into an aged bitumen sample in 50 min.

Hurricane María drives increased indoor proliferation of filamentous fungi in San Juan, Puerto Rico: a two-year culture-based approach.

Extensive flooding caused by Hurricane María in Puerto Rico (PR) created favorable conditions for indoor growth of filamentous fungi. These conditions represent a public health concern as contamination by environmental fungi is associated with a higher prevalence of inflammatory respiratory conditions. This work compares culturable fungal spore communities present in homes that sustained water damage after Hurricane María to those present in dry, non-flooded homes. We collected air samples from 50 houses in a neighborhood in San Juan, PR, 12 and 22 months after Hurricane María. Self-reported data was used to classify the homes as flooded, water-damage or dry non-flooded. Fungi abundances, composition and diversity were analyzed by culturing on two media. Our results showed no significant differences in indoor fungal concentrations (CFU/m3) one year after the Hurricane in both culture media studied (MEA and G25N). During the second sampling period fungal levels were 2.7 times higher in previously flooded homes (Median = 758) when compared to dry homes (Median = 283), (p-value < 0.005). Fungal profiles showed enrichment of Aspergillus species inside flooded homes compared to outdoor samples during the first sampling period (FDR-adjusted p-value = 0.05). In contrast, 22 months after the storm, indoor fungal composition consisted primarily of non-sporulated fungi, most likely basidiospores, which are characteristic of the outdoor air in PR. Together, this data highlights that homes that suffered water damage not only have higher indoor proliferation of filamentous fungi, but their indoor fungal populations change over time following the Hurricane. Ultimately, after nearly two years, indoor and outdoor fungal communities converged in this sample of naturally ventilated homes.

Sporicidal mechanism of the combination of ortho-phthalaldehyde and benzyldimethyldodecylammonium chloride as a disinfectant against the Bacillus subtilis spores.

Previous studies have shown that the combination disinfectant, Ortho-phthalaldehyde and benzyldimethyldodecylammonium chloride (ODB), can effectively kill a variety of microorganisms, such as Escherichia coli, Staphylococcus aureus, and Candida albicans. To observe the sporicidal ability and mechanism of ODB for spores, Bacillus subtilis spores were used as the research object in this experiment. TEM images revealed that ODB destroyed the integrity of the coat, cortex, and inner membrane of the spores after 0.5-h treatment, and the nuclear material was also broken and exuded after 4-h treatment. The broken structure led to the release of dipicolinic acid (DPA) in large amount. The results show that B. subtilis spores can be effetely killed by ODB through destroying the structure of the spores.

Morphological, ultrastructural, and molecular analysis of a new species of Myxobolus (Cnidaria, Myxosporea) parasitizing Apareiodon piracicabae (Characiformes, Parodontidae) from Brazil.

About forty-two species of Myxobolus have been previously described to parasitize characiform fishes in South America. Here, we described a new myxozoan species, Myxobolus parodontidis n. sp., in the gills of Apareiodon piracicabae (Characiformes, Parodontidae) from the streams of the Middle Paranapanema River basin, Brazil. The proposed new species is supported by a combination of morphological, ultrastructural, and molecular characterization (small subunit ribosomal DNA). Thirteen specimens of A. piracicabae were analyzed and 30.8% was infected by Myxobolus parodontidis n. sp. The myxospores was classified as intralamellar asymmetric type. A few aberrant myxospores with three polar capsules were observed: the spore length and width were the same of normal myxospores, but the polar capsules had smaller sizes. Ultrastructural analysis showed that the plasmodial membrane of Myxobolus parodontidis n. sp. was in direct contact with the host tissue and a connective capsule surrounding the plasmodium was not observed. The phylogenetic analysis showed that the main influence in the clustering of species of myxobolids seems to be related to the phylogenetic relationships established among fish hosts, mainly at the level of family and order. This is the first record of a myxozoan species parasitizing parodontid fish, contributing to the knowledge of the biodiversity of myxozoans from Brazil.

Simulation of thermal sanitization of air with heat recovery as applied to airborne pathogen deactivation.

The technique of air sterilization by thermal effect was revisited in this work. The impact of incorporating a high efficiency heat recovery exchanger to a sterilizing cell was especially assessed. A mathematical model was developed to study the dynamics and the steady state of the sterilizer. Computer simulation and reported data of thermal inactivation of pathogens permitted obtaining results for a proof-of-concept. The simulation results confirmed that the incorporation of a heat recovery exchanger permits saving more than 90% of the energy needed to heat the air to the temperature necessary for sterilization, i.e., sterilization without heat recovery consumes 10-20 times the energy of the same sterilization device with heat recovery. Sanitization temperature is the main process variable for sanitization, a fact related to the activated nature of the thermal inactivation of viruses and bacteria. Heat recovery efficiency was a strong function of the heat transfer parameters but also rather insensitive to the cell temperature. The heat transfer area determined the maximum capacity of the sterilizer (maximum air flowrate) given the restrictions of minimum sanitization efficiency and maximum power consumption. The proposed thermal sterilization device has important advantages of robustness and simplicity over other commercial sterilization devices, needing practically no maintenance and eliminating a big variety of microorganisms to any desired degree. For most pathogens, the inactivation can be total. This result is not affected by the uncertainties in thermal inactivation data, due to the Arrhenius-like dependence of inactivation. Temperature can be adjusted to achieve any removal degree.

Effect of bactofugation of raw milk on total bacterial and aerobic spore counts and on microbial diversity of sporeformers / Efeito da bactofugação do leite cru nas contagens bacteriana total, esporos aeróbios e na diversidade microbiana de formadores de esporos

This study describes the effect of bactofugation (10,000 × g in a continuous flow of 10,000 L/h) of three batches of raw milk on total bacterial count (TBC) and aerobic spore count, and it also shows the effect on the microbial diversity of spore-forming bacteria by analysing their genetic variability through molecular approaches. Given that milk must be preheated to approximately 55 °C before being bactofuged, for comparison, the three batches were evaluated at different stages as refrigerated raw, preheated, and bactofuged milk. For preheated milk, it was found that bactofugation caused a significant reduction (p < 0.05) of 99.52% (from 4.5 × 106 to 2.1 × 104 CFU/mL) in the mean TBC and of 95% (from 333 to 17 CFU/mL) in the aerobic mesophilic spore count. Due to the effect of bactofugation on preheated milk, a reduction of 82% was observed in both TBC and aerobic spore count. With respect to diversity, 107 isolates from raw milk, prior to bactofugation, and 16 isolates from bactofuged milk were recovered and grouped into 40 and 8 clusters, respectively. The predominant species detected in raw and preheated milk were Bacillus toyonensis (63% - 20 clusters) and Lysinibacillus fusiformis (15% - 8 clusters). Proportionally, B. toyonensis (69% - 6 clusters) and L. fusiformis (25% - 1 cluster) were predominant in bactofuged milk. B. pumilus, L. varians, B. flexus, B. invictae, and B. megaterium, bacteria with a known milk spoilage potential, were isolated from milk prior to bactofugation, and they reduced to undetectable levels in bactofuged milk. Bactofugation of milk, therefore, reduces the TBC and aerobic spore count, with a significant effect in reducing the microbial diversity of spore-forming bacteria, proportional to their incidence in raw milk. Therefore, bactofugation can be an alternative to increase the shelf life and technological potential of milk.(AU)

Esse estudo descreve o efeito da bactofugação (10,000 × g em fluxo contínuo de 10,000 L/h) de três lotes de leite cru nas contagens bacterianas totais (CBT) e de esporos aeróbios, verificando também o efeito sobre a diversidade microbiana dos formadores de esporos por abordagem molecular de variabilidade genética. Como o leite a ser bactofugado deve ser pré-aquecido (≈55ºC), os três lotes foram avaliados enquanto cru refrigerado, pré-aquecido e bactofugado. Em associação com o pré-aquecimento, foi verificado que a bactofugação promoveu a redução significativa (p < 0.05) de 99,52% (4,5 x 106 para 2,1 x 104 UFC/mL) na contagem bacteriana total e 95% (333 para 17 UFC/mL) dos esporos aeróbios mesófilos. Considerando o efeito isolado da bactofugação sobre o leite já pré-aquecido, foi observada a redução de 82% tanto para CBT quanto para formadores de esporos. Em relação à diversidade, foram recuperados 107 isolados do leite anterior à bactofugação e 16 isolados do leite bactofugado, agrupados em 40 e 8 clusters, respectivamente. Foi observado predomínio das espécies Bacillus toyonensis (63% - 20 clusters) e Lysinibacillus fusiformis (15% - 8 clusters) no leite cru e pré-aquecido e, proporcionalmente, B. toyonensis (69% - 6 clusters) e L. fusiformis (25% - 1 cluster) no leite bactofugado. Bacillus pumilus, Lysinibacillus varians, B. flexus, B. invictae, e B. megaterium, isolados do leite antes da bactofugação e com potencial deteriorante conhecido, foram reduzidos a níveis indetectáveis no leite bactofugado. A bactofugação do leite, portanto, reduz a CBT e as contagens de esporos aeróbios, também com efeito significativo na redução da diversidade de formadores de esporos, proporcionalmente, conforme a sua incidência no leite cru, tendo potencial para ser utilizado como alternativa para aumento da vida útil e potencial tecnológico do leite.(AU)