RESUMEN
Laboratory pasteurization count (LPC) enumerates thermoduric bacteria and is one parameter used to assess raw milk quality. No regulatory limit has presently been set for LPC, but LPC data are used by some dairy processors and cooperatives to designate raw milk quality premiums paid to farmers and may also be used for troubleshooting bacterial contamination issues. Although it is occasionally used as a proxy for levels of bacterial spores in raw milk, limited knowledge is available on the types of organisms that are enumerated by LPC in contemporary raw milk supplies. Although historical studies have reported that thermoduric bacteria quantified by LPC may predominantly represent gram-positive cocci, updated knowledge on microbial populations enumerated by LPC in contemporary organic raw milk supplies is needed. To address this gap, organic raw milk samples from across the United States (n = 94) were assessed using LPC, and bacterial isolates were characterized. LPC ranged from below detection (<0.70 log cfu/mL) to 4.07 log cfu/mL, with a geometric mean of 1.48 log cfu/mL. Among 380 isolates characterized by 16S rDNA sequencing, 52.6%, 44.5%, and 2.4% were identified as gram-positive sporeformers, gram-positive nonsporeformers, and gram-negatives, respectively; 0.5% could not be categorized into those groups because they could only be assigned a higher level of taxonomy. Isolates identified as gram-positive sporeformers were predominantly Bacillus (168/200), and gram-positive nonsporeformers were predominantly Brachybacterium (56/169) and Kocuria (47/169). To elucidate if the LPC level can be an indicator of the type of thermoduric (e.g., sporeforming bacteria) present in raw milk, we evaluated the proportion of sporeformers in raw milk samples with LPC of ≤100 cfu/mL, 100 to 200 cfu/mL, and ≥200 cfu/mL (51%, 67%, and 35%), showing a trend for sporeformers to represent a smaller proportion of the total thermoduric population when LPC increases, although overall linear regression showed no significant association between the proportion of sporeformers and the LPC concentration. Hence, LPC level alone provides no insight into the makeup of the thermoduric population in raw milk, and further characterization is needed to elucidate the bacterial drivers of elevated LPC in raw milk. We therefore further characterized the isolates from this study using MALDI-TOF mass spectrometry (MALDI-TOF MS), a rapid microbial identification tool that is more readily available to dairy producers than 16S rDNA PCR and sequencing. Although our data indicated agreement between 16S rDNA sequencing and MALDI-TOF MS for 66.6% of isolates at the genus level, 24.2% and 9.2% could not be reliably identified or were mischaracterized using MALDI-TOF MS, respectively. This suggests that further optimization of this method is needed to allow for accurate characterization of thermoduric organisms commonly found in raw milk. Ultimately, our study provides a contemporary perspective on thermoduric bacteria selected by the LPC method and establishes that the LPC alone is not sufficient for identifying the bacterial drivers of LPC levels. Further development of rapid characterization methods that are accessible to producers, cooperatives, and processors will support milk quality troubleshooting efforts and ultimately improve outcomes for dairy industry community members.
Asunto(s)
Leche , Pasteurización , Esporas Bacterianas , Leche/microbiología , Animales , Esporas Bacterianas/aislamiento & purificación , Recuento de Colonia MicrobianaRESUMEN
A microbiological study was conducted on 41 insect product samples (29 raw frozen [21 domestic and 8 imported], 10 powdered, and 2 processed), which were commercially available in Japan. The total aerobic count for raw frozen insects was 5.61 log cfu/g (range: 2.52-8.40), whereas the powdered insect count was 2.89 log cfu/g (range: 1.00-4.57). The bacterial count was significantly higher in raw frozen insects (p < 0.05). The coliform count for the raw frozen insects ranged from <1 to 6.90 log cfu/g, and that for the powdered insects ranged from <1 to 1.00 log cfu/g. The number of samples with values above the detection limit was significantly higher in raw frozen insects (p < 0.05). The detection frequencies of aerobic spores (<1-4.63 log cfu/g), anaerobic spores (<0-4.40 log cfu/g), and Bacillus cereus (<1.7-3.83 log cfu/g) showed no sample type-related significant difference. Listeria spp. was isolated from four samples of raw frozen insects, one of which was Listeria monocytogenes. We did not detect any of the following: Salmonella spp., Shiga toxin-producing E. coli (STEC), Campylobacter jejuni/coli, or pathogenic Yersinia. We isolated insect products retailed in Japan harboring food poisoning bacteria, including L. monocytogenes and B. cereus. In particular, raw frozen products displayed high levels of hygienic indicator bacteria.
Asunto(s)
Microbiología de Alimentos , Listeria monocytogenes , Japón , Animales , Listeria monocytogenes/aislamiento & purificación , Recuento de Colonia Microbiana , Bacillus cereus/aislamiento & purificación , Esporas Bacterianas/aislamiento & purificación , Contaminación de Alimentos/análisis , Insectos/microbiología , Alimentos Congelados/microbiología , Insectos Comestibles/microbiología , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Salmonella/aislamiento & purificaciónRESUMEN
Many bacterial species are capable of forming long-lived dormant cells. The best characterized are heat and desiccation resistant spores produced by many Gram-positive species. Less characterized are dormant cysts produced by several Gram-negative species that are somewhat tolerant to increased temperature and very resistant to desiccation. While there is progress in understanding regulatory circuits that control spore germination, there is scarce information on how Gram-negative organisms emerges from dormancy. In this study, we show that R. centenum cysts germinate by emerging a pair of motile vegetative cells from a thick cyst cell wall coat ~ 6 hrs post induction of germination. Time-lapse transcriptomic analysis reveals that there is a defined temporal pattern of gene expression changes during R. centenum cyst germination. The first observable changes are increases in expression of genes for protein synthesis, an increase in expression of genes involved in the generation of a membrane potential and the use of this potential for ATP synthesis via ATPase expression. These early events are followed by expression changes that affect the cell wall and membrane composition, followed by expression changes that promote chromosome replication. Midway through germination, expression changes occur that promote the flow of carbon through the TCA cycle to generate reducing power and parallel synthesis of electron transfer components involved in oxidative phosphorylation. Finally, late expression changes promote the synthesis of a photosystem as well as flagellar and chemotaxis components for motility.
Asunto(s)
Rhodospirillum centenum/genética , Rhodospirillum centenum/metabolismo , Esporas Bacterianas/genética , Pared Celular/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación Bacteriana de la Expresión Génica/genética , Biosíntesis de Proteínas/genética , Esporas/genética , Esporas/aislamiento & purificación , Esporas Bacterianas/aislamiento & purificación , Esporas Bacterianas/metabolismo , Transcriptoma/genéticaRESUMEN
We describe herein a simple procedure for quantifying endospore abundances in ancient and organic-rich permafrost. We repeatedly (10x) extracted and fractionated permafrost using a tandem filter assembly composed of 3 and 0.2 µm filters. Then, the 0.2 µm filter was washed (7x), autoclaved, and the contents eluted, including dipicolinic acid (DPA). Time-resolved luminescence using Tb(EDTA) yielded a LOD of 1.46 nM DPA (6.55 × 103 endospores/mL). In review, DPA/endospore abundances were ~2.2-fold greater in older 33 ky permafrost (258 ± 36 pmol DPA gdw-1; 1.15 × 106 ± 0.16 × 106 spores gdw-1) versus younger 19 ky permafrost (p = 0.007297). This suggests that dormancy increases with permafrost age.
Asunto(s)
Hielos Perennes/química , Espectrometría de Fluorescencia/métodos , Esporas Bacterianas/química , Esporas Bacterianas/aislamiento & purificación , Quelantes/análisis , Quelantes/química , Quelantes/aislamiento & purificación , Ácidos Picolínicos/análisis , Ácidos Picolínicos/química , Ácidos Picolínicos/aislamiento & purificación , Terbio/químicaRESUMEN
AIM: Rapid detection of biological agents in biodefense is critical for operational, tactical and strategic levels as well as for medical countermeasures. Yersinia pestis, Francisella tularensis, and Bacillus anthracis are high priority agents of biological warfare or bioterrorism and many response forces use lateral flow assays (LFAs) for their detection. Several companies produce these assays, which offer results in short time and are easy to use. Despite their importance, only few publications on the limits of detection (LOD) for LFAs are available. Most of these studies used inactivated bacteria or risk group-2 strains. As the inactivation process in previous studies might have affected the tests' performances, it was our aim in this study to determine and compare the LOD of several commercially available LFAs using viable risk group-3 strains. METHODS AND RESULTS: Lateral flow assays from four different companies for the detection of following bacteria were evaluated: Y. pestis, F. tularensis and B. anthracis spores. Two independent quantification methods for each target organism were applied, in order to ensure high quantification accuracy. LODs varied greatly between tests and organisms and ranged between 104 for Y. pestis-tests and as high as >109 for one B. anthracis-test. CONCLUSION: This work precisely determined the LODs of LFAs from four commercial suppliers. The herein determined LODs differed from results of previous studies. This illustrates the need for using accurately quantified viable risk group 3-strains for determining such LODs. SIGNIFICANCE AND IMPACT OF THE STUDY: Our work bridges an important knowledge gap with regard to LFA LOD. The LODs determined in this study will facilitate better assessment of LFA-results. They illustrate that a negative LFA result is not suited to exclude the presence of the respective agent in the analyzed sample.
Asunto(s)
Bacillus anthracis/aislamiento & purificación , Técnicas Bacteriológicas/métodos , Francisella tularensis/aislamiento & purificación , Inmunoensayo/métodos , Yersinia pestis/aislamiento & purificación , Infecciones Bacterianas/diagnóstico , Infecciones Bacterianas/microbiología , Humanos , Límite de Detección , Viabilidad Microbiana , Esporas Bacterianas/aislamiento & purificaciónRESUMEN
The post-glacial Baltic Sea has experienced extreme changes that are archived today in the deep sediments. IODP Expedition 347 retrieved cores down to 100 m depth and studied the climate history and the deep biosphere. We here review the biogeochemical and microbiological highlights and integrate these with other studies from the Baltic seabed. Cell numbers, endospore abundance and organic matter mineralization rates are extremely high. A 100-fold drop in cell numbers with depth results from a small difference between growth and mortality in the ageing sediment. Evidence for growth derives from a D:L amino acid racemization model, while evidence for mortality derives from the abundance and potential activity of lytic viruses. The deep communities assemble at the bottom of the bioturbated zone from the founding surface community by selection of organisms suited for life under deep sediment conditions. The mean catabolic per-cell rate of microorganisms drops steeply with depth to a life in slow-motion, typical for the deep biosphere. The subsurface life under extreme energy limitation is facilitated by exploitation of recalcitrant substrates, by biochemical protection of nucleic acids and proteins and by repair mechanisms for random mismatches in DNA or damaged amino acids in proteins.
Asunto(s)
Bacterias/clasificación , Sedimentos Geológicos/microbiología , Virus/clasificación , Bacterias/genética , Países Bálticos , Océanos y Mares , Esporas Bacterianas/aislamiento & purificación , Virus/genéticaRESUMEN
Bacillus cereus is a ubiquitous endospore-forming bacterium, which mainly affects humans as a food-borne pathogen. Bacillus cereus can contaminate groundwater used to irrigate food crops. Here, we examined the ability of the emetic strain B. cereus F4810/72 to survive abiotic conditions encountered in groundwater. Our results showed that vegetative B. cereus cells rapidly evolved in a mixed population composed of endospores and asporogenic variants bearing spo0A mutations. One asporogenic variant, VAR-F48, was isolated and characterized. VAR-F48 can survive in sterilized groundwater over a long period in a vegetative form and has a competitive advantage compared to its parental strain. Proteomics analysis allowed us to quantify changes to cellular and exoproteins after 24 and 72 h incubation in groundwater, for VAR-F48 compared to its parental strain. The results revealed a significant re-routing of the metabolism in the absence of Spo0A. We concluded that VAR-F48 maximizes its energy use to deal with oligotrophy, and the emergence of spo0A-mutated variants may contribute to the persistence of emetic B. cereus in natural oligotrophic environments.
Asunto(s)
Bacillus cereus/fisiología , Proteínas Bacterianas/genética , Enfermedades Transmitidas por los Alimentos/microbiología , Agua Subterránea/microbiología , Factores de Transcripción/genética , Bacillus cereus/genética , Bacillus cereus/aislamiento & purificación , Bacillus cereus/metabolismo , Proteínas Bacterianas/metabolismo , Microbiología de Alimentos , Humanos , Viabilidad Microbiana/genética , Mutación , Esporas Bacterianas/genética , Esporas Bacterianas/aislamiento & purificación , Esporas Bacterianas/metabolismo , Esporas Bacterianas/fisiología , Factores de Transcripción/metabolismoRESUMEN
The acquisition of the infant gut microbiota is key to establishing a host-microbiota symbiosis. Microbially produced metabolites tightly interact with the immune system, and the fermentation-derived short-chain fatty acid butyrate is considered an important mediator linked to chronic diseases later in life. The intestinal butyrate-forming bacterial population is taxonomically and functionally diverse and includes endospore formers with high transmission potential. Succession, and contribution of butyrate-producing taxa during infant gut microbiota development have been little investigated. We determined the abundance of major butyrate-forming groups and fermentation metabolites in faeces, isolated, cultivated and characterized the heat-resistant cell population, which included endospores, and compared butyrate formation efficiency of representative taxa in batch cultures. The endospore community contributed about 0.001% to total cells, and was mainly composed of the pioneer butyrate-producing Clostridium sensu stricto. We observed an increase in abundance of Faecalibacterium prausnitzii, butyrate-producing Lachnospiraceae and faecal butyrate levels with age that is likely explained by higher butyrate production capacity of contributing taxa compared with Clostridium sensu stricto. Our data suggest that a successional arrangement and an overall increase in abundance of butyrate forming populations occur during the first year of life, which is associated with an increase of intestinal butyrate formation capacity.
Asunto(s)
Bacterias/aislamiento & purificación , Bacterias/metabolismo , Butiratos/metabolismo , Microbioma Gastrointestinal/fisiología , Bacterias/clasificación , Bacterias/genética , Ácidos Grasos Volátiles/metabolismo , Heces/química , Heces/microbiología , Fermentación , Humanos , Lactante , Intestinos/crecimiento & desarrollo , Intestinos/microbiología , Esporas Bacterianas/clasificación , Esporas Bacterianas/genética , Esporas Bacterianas/aislamiento & purificación , Esporas Bacterianas/metabolismoRESUMEN
Clostridium botulinum is a significant food safety concern due to its ability to produce highly potent neurotoxin and resistant endospores. Vegetarian sausages have become a popular source of plant protein and alternative for meat products. While vegetarian sausages have not been linked to botulism, numerous outbreaks due to preserved vegetables suggest a frequent occurrence of C. botulinum spores in the raw material. The product formulation of vegetarian sausages involves limited NaCl and preservatives, and shelf-lives may be several months. The safety of vegetarian sausages thus relies mainly on heat treatment and chilled storage. The main food safety concern is C. botulinum Group II that can grow and produce toxin at refrigeration temperatures. Here we show a high overall prevalence (32%) of C. botulinum in 74 samples of vegetarian sausages from seven producers. Both Groups I and II strains and genes for neurotoxin types A, B, E and F were detected in the products. The highest cell counts (1200 spores/kg) were observed for C. botulinum Group II in products with remaining shelf-lives of 6 months at the time of purchase. We conclude that vacuum-packaged vegetarian sausage products frequently contain C. botulinum spores and may possess a high risk of C. botulinum growth and toxin production. Chilled storage below 3°C and thorough reheating before consumption are warranted.
Asunto(s)
Clostridium botulinum/aislamiento & purificación , Alimentos en Conserva/microbiología , Verduras/microbiología , Toxinas Botulínicas/genética , Clostridium botulinum/clasificación , Clostridium botulinum/genética , Clostridium botulinum/crecimiento & desarrollo , Recuento de Colonia Microbiana , Seguridad de Productos para el Consumidor , Manipulación de Alimentos/métodos , Microbiología de Alimentos , Genotipo , Esporas Bacterianas/clasificación , Esporas Bacterianas/genética , Esporas Bacterianas/crecimiento & desarrollo , Esporas Bacterianas/aislamiento & purificación , VegetarianosRESUMEN
The safety of current UK industry practice (including shelf-life) for chilled, vacuum/modified atmosphere-packed fresh red meat (beef, lamb and pork) held at 3°C-8°C has been evaluated with respect to non-proteolytic Clostridium botulinum. UK industry typically applies a retail pack shelf-life at 3°C-8°C to 13 days for fresh red meat, with a maximum of 23 days for beef, 27 days for lamb, and 18 days for pork. An exposure assessment established that current commercial practice for fresh red meat provided strong protection with more than 1010 person servings marketed in the UK without association with foodborne botulism. A challenge test demonstrated that spores of non-proteolytic C. botulinum inoculated on chilled vacuum-packed fresh red meat did not lead to detectable neurotoxin at day 50 for beef, day 35 for lamb, or day 25 for pork (i.e. <40 pg type B toxin and type E toxin g-1 of meat). The products were visually spoiled many days before these end points. The exposure assessment and challenge test demonstrated the safety of current UK industry practices for the shelf-life of fresh, vacuum-packed beef, lamb and pork held at 3°C-8°C with respect to C. botulinum, and that botulinum neurotoxin was not detected within their organoleptic shelf-life.
Asunto(s)
Botulismo/epidemiología , Exposición Dietética/estadística & datos numéricos , Embalaje de Alimentos/métodos , Almacenamiento de Alimentos/métodos , Carne Roja/microbiología , Animales , Atmósfera , Botulismo/microbiología , Bovinos , Clostridium botulinum/aislamiento & purificación , Frío , Exposición Dietética/análisis , Microbiología de Alimentos , Incidencia , Neurotoxinas/análisis , Carne Roja/análisis , Medición de Riesgo , Ovinos , Olfato , Esporas Bacterianas/aislamiento & purificación , Porcinos , Gusto , VacioRESUMEN
The effects of chitosan with 95% deacetylation degree (DD95) on the spore germination, cell proliferation, and heat resistance of Clostridium perfringens CCRC 10,648 and CCRC 13,019 were investigated, and its application on pork sausage with sodium nitrite reduction was also evaluated. DD95 chitosan can strongly reduce the heat resistance of both strains. The D80 and D100 values for strain CCRC 13,019 decreased from 40.98 and 4.64 min to 39.21 and 3.26 min, respectively, as a result of adding 250 ppm DD95; meanwhile, addition of chitosan decreased the D80 and D100 values for CCRC 10,648 from 41.15 and 6.46 min to 39.52 and 3.78 min, respectively. In pork sausage, addition of 3000 ppm DD95 chitosan considerably slowed down the bacterial proliferation and volatile basic nitrogen production. There were no significant differences in color (L* and b* values), shearing force, and hardness in the pork sausages with or without DD95 chitosan during storage at 4 and 25 °C. However, the addition of DD95 chitosan in pork sausage significantly retarded the decrease of the a* value. Therefore, DD95 chitosan could reduce the concentration of sodium nitrite required in pork sausages for color retention.
Asunto(s)
Quitosano/administración & dosificación , Infecciones por Clostridium/prevención & control , Clostridium perfringens/efectos de los fármacos , Conservantes de Alimentos/administración & dosificación , Enfermedades Transmitidas por los Alimentos/prevención & control , Productos de la Carne/microbiología , Animales , Proliferación Celular/efectos de los fármacos , Quitosano/aislamiento & purificación , Infecciones por Clostridium/microbiología , Clostridium perfringens/aislamiento & purificación , Crustáceos/química , Conservación de Alimentos/métodos , Conservantes de Alimentos/aislamiento & purificación , Enfermedades Transmitidas por los Alimentos/microbiología , Respuesta al Choque Térmico/efectos de los fármacos , Humanos , Nitrito de Sodio/administración & dosificación , Esporas Bacterianas/aislamiento & purificación , PorcinosRESUMEN
This study aims to characterize Bacillus subtilis complex group from raw, pasteurized, and packaged extended shelf-life (ESL) milk samples, to determine their biofilm potential and source-track the microbial contaminants to control their presence during processing. Isolates were characterized using multi-locus sequence typing (MLST) with 7 housekeeping genes. The primers used were designed from the coding regions with the highest number of polymorphic sites. The heat resistance profile indicated that all 12 isolates are psychrotolerant as well as thermophilic, with temperature ranges of 6°C to 55°C (B43, B44, B52, B54, B55, B56, B57), 6°C to 60°C (B46, B47, B48), and 15°C to 60°C (B49, B50). A general linear model 2-way repeated-measure ANOVA of the biofilm-forming potential of the isolates shows a statistically significant difference across the time of incubation (6, 12, 18, and 24 h). All isolates except 2 formed moderate to strong biofilms, with B44 having the most robust biofilm formation (3.14 ± 0.60). Scanning electron and confocal microscopy images reveal the strain specificity of the biofilm structure. The MLST analysis identified all isolates as belonging to either B. subtilis or Bacillus velezensis. All the isolates were novel sequence types (ST) when compared with the PubMLST database (https://pubmlst.org/) but showed relatedness to isolates in the raw milk that was processed. The closest ST are 96 for B. velezensis and 128 for B. subtilis, mostly isolated from soil. This study presents the significance of biofilms of thermophilic B. subtilis and B. velezensis and their possible perpetuation in the dairy processing plant. The information provided is a call for an innovative food contact surface or any other intervention that can minimize or prevent microbial adhesion in the processing plant, to prevent negative effects in ESL milk.
Asunto(s)
Bacillus subtilis/crecimiento & desarrollo , Bacillus/crecimiento & desarrollo , Biopelículas , Industria Lechera , Leche/microbiología , Animales , Bacillus/aislamiento & purificación , Tipificación de Secuencias Multilocus , Pasteurización , Esporas Bacterianas/aislamiento & purificaciónRESUMEN
One of the most severe quality defects in hard and semi-hard cheese, the late blowing defect, is caused by endospore-forming bacteria of the genus Clostridium. To minimize financial losses and waste of resources due to cheese spoilage, raw milk with elevated clostridial spore counts should not be used for the production of certain cheese types. In this context, threshold values of clostridial spore concentrations that cause quality defects in cheese are still under debate. To improve our understanding about late blowing defects, further information on the correlation between clostridial spore concentrations in milk and cheese quality is indispensable. Thus, the aim of this study was to monitor the microbiological quality of milk used for Alpine cheese production regarding clostridial endospore levels to facilitate the establishment of threshold spore concentrations that guarantee the absence of quality defects in Austrian cheese. For this purpose, we monitored clostridial endospore levels in vat milk of 4 Alpine dairies throughout the summer grazing period in 2018. Surprisingly, we observed almost complete absence of butyric acid-producing clostridia in milk and no blowing defects in cheese. Hence, critical clostridial spore concentrations could not be verified. Moreover, the observed low spore levels reveal that the prohibition of silage feeding and good farming practices effectively minimize clostridial endospore counts in milk and ensure the manufacture of high-quality cheese even if technological possibilities are limited.
Asunto(s)
Queso/microbiología , Clostridium/aislamiento & purificación , Leche/microbiología , Esporas Bacterianas/aislamiento & purificación , Animales , Austria , Recuento de Colonia Microbiana , Microbiología de AlimentosRESUMEN
Infections linked to Clostridium difficile are a significant cause of suffering. In hospitals, the organism is primarily acquired through the faecal-oral route as spores excreted by infected patients contaminate the healthcare environment. We previously reported that members of the C. difficile group varied widely in their ability to adhere to stainless steel and proposed that these differences were a consequence of variations in spore architecture. In this study of clinical isolates and spore coat protein mutants of C. difficile we identified three distinct spore surfaces morphotypes; smooth, bag-like and "pineapple-like" using scanning electron microscopy (SEM). The frequency of each morphotype in a spore population derived from a single isolate varied depending on the host strain and the method used to produce and purify the spores. Our results suggest that the inclusion of a sonication step in the purification process had a marked effect on spore structure. In an attempt to link differences in spore appearance with key structural spore proteins we compared the morphology of spores of CD630 to those produced by CD630 variants lacking either CotE or BclA. While SEM images revealed no obvious structural differences between CD630 and its mutants we did observe significant differences (pâ¯<â¯0.001) in relative hydrophobicity suggesting that modifications had occurred but not at a level to be detectable by SEM. In conclusion, we observed significant variation in the spore morphology of clinical isolates of C. difficile due in part to the methods used to produce them. Sonication in particular can markedly change spore appearance and properties. The results of this study highlight the importance of adopting "standard" methods when attempting to compare results between studies and to understand the significance of their differences.
Asunto(s)
Clostridioides difficile/citología , Clostridioides difficile/ultraestructura , Esporas Bacterianas/citología , Esporas Bacterianas/ultraestructura , Pared Celular/ultraestructura , Clostridioides difficile/clasificación , Clostridioides difficile/aislamiento & purificación , Interacciones Hidrofóbicas e Hidrofílicas , Especificidad de la Especie , Esporas Bacterianas/aislamiento & purificación , Propiedades de SuperficieRESUMEN
A colorimetric polydiacetylene (PDA) paper strip sensor that can specifically recognize Bacillus thuringiensis (BT) HD-73 spores is described in this work. The target-specific aptamer was combined with PDA, and the aptamer-conjugated PDA vesicles were then coated on polyvinylidene fluoride (PVDF) paper strips by a simple solvent evaporation method. The PDA-aptamer paper strips can be used to detect the target without any pre-treatment. Using the paper strip, the presence of BT spores is directly observable by the naked eye based on the unique blue-to-red color transition of the PDA. Quantitative studies using the paper strip were also carried out by analyzing the color transitions of the PDA. The specificity of this PDA sensor was verified with a high concentration of Escherichia coli, and no discernable change was observed. The observable color change in the paper strip occurs in less than 1 h, and the limit of detection is 3 × 107 CFU/mL, much below the level harmful to humans. The PDA-based paper sensor, developed in this work, does not require a separate power or detection device, making the sensor strip highly transportable and suitable for spore analysis anytime and anywhere. Moreover, this paper sensor platform is easily fabricated, can be adapted to other targets, is highly portable, and is highly specific for the detection of BT spores.
Asunto(s)
Bacillus thuringiensis/aislamiento & purificación , Técnicas Biosensibles , Colorimetría , Esporas Bacterianas/aislamiento & purificación , Polímero PoliacetilénicoRESUMEN
The phylum Firmicutes comprises seven classes where most species are either aerobic or anaerobic endospore former. Inside Firmicutes, species allocated in the genus Bacillus and related genera are collectively named aerobic endospore-forming bacteria (AEFB), and the soil is their major reservoir. AEFB have great importance in health, agriculture, and biotechnology although the more studied species are Bacillus subtilis and the human pathogens Bacillus cereus and Bacillus anthracis. AEFB have great importance in health, agriculture, and biotechnology; although the knowledge about these organisms is based on few species, notably, Bacillus subtilis, Bacillus cereus, and Bacillus anthracis. In this work, we generated partial 16S rRNA gene sequences of both strands of 192 AEFB strains isolated from soils of Distrito Federal, Brazil (SDF strains). The resulting consensus sequences were used to obtain taxonomic assignment and establish the phylogenetic relationships among these strains. Through this approach, we could observe that classified SDF strains were distributed among genera Bacillus (169 strains; 88.02%), Paenibacillus (11; 5.73%), Lysinibacillus (6; 3.13%), Brevibacillus (4; 2.08%), Terribacillus (1; 0.52%), and Rummeliibacillus (1; 0.52%). Phylogenetic trees revealed these 192 SDF strains can be segregated into eight groups spanning families Bacillaceae and Paenibacillaceae belonging to the order Bacillales. To expand the knowledge about the diversity of these SDF strains, further studies regarding characterization with different methodologies are underway.
Asunto(s)
Bacillales/clasificación , Bacillales/aislamiento & purificación , Filogenia , Microbiología del Suelo , Bacillales/genética , Brasil , ADN Bacteriano/genética , Variación Genética , ARN Ribosómico 16S/genética , Esporas Bacterianas/clasificación , Esporas Bacterianas/genética , Esporas Bacterianas/aislamiento & purificaciónRESUMEN
The purpose of this article is to highlight some areas of research with spores of bacteria of Firmicute species in which the methodology too commonly used is not optimal and generates misleading results. As a consequence, conclusions drawn from data obtained are often flawed or not appropriate. Topics covered in the article include the following: (i) the importance of using well-purified bacterial spores in studies on spore resistance, composition, killing, disinfection and germination; (ii) methods for obtaining good purification of spores of various species; (iii) appropriate experimental approaches to determine mechanisms of spore resistance and spore killing by a variety of agents, as well as known mechanisms of spore resistance and killing; (iv) common errors made in drawing conclusions about spore killing by various agents, including failure to neutralize chemical agents before plating for viable spore enumeration, and equating correlations between changes in spore properties accompanying spore killing with causation. It is hoped that a consideration of these topics will improve the quality of spore research going forward.
Asunto(s)
Bacillales/efectos de los fármacos , Clostridiales/efectos de los fármacos , Esporas Bacterianas/efectos de los fármacos , Farmacorresistencia Bacteriana , Esporas Bacterianas/aislamiento & purificaciónRESUMEN
AIMS: To develop a gel formulation to trigger a visual signal for rapid disclosure of the location and extent of surface contamination with viable Bacillus anthracis spores. METHODS AND RESULTS: Methylumbelliferyl-α-d-glucopyranoside was combined with hyaluronic acid to produce a gel that could be applied to a surface as a coating. It remained hydrated for a sufficient time for α-glucosidase activity present in intact B. anthracis spores to cleave the substrate and release the fluorescent product, methylumbelliferone. The presence of B. anthracis spores could be disclosed at 5 × 104 CFU per reaction test well (0·32 cm2 ) both visually and using fluorescence detection equipment. CONCLUSIONS: The disclosure gel provides a rapid, visual response to the presence of B. anthracis spores on a surface. SIGNIFICANCE AND IMPACT OF THE STUDY: The disclosure gel demonstrates the first steps towards the development of a formulation that can provide nonspecialist users with a visual alert to the presence of B. anthracis spores on a surface. It is envisioned that such a formulation would be beneficial in scenarios where exposure to spore release is a risk, and could be used in the initial assessment of equipment to aid prioritization and localized execution of a decontamination strategy.
Asunto(s)
Bacillus anthracis/aislamiento & purificación , Descontaminación/métodos , Exposición a Riesgos Ambientales/prevención & control , Técnicas Microbiológicas/métodos , Esporas Bacterianas/aislamiento & purificación , Bacillus anthracis/enzimología , Bacillus anthracis/metabolismo , Ácido Hialurónico/química , Himecromona/química , Himecromona/metabolismo , Indicadores y Reactivos , Esporas Bacterianas/enzimología , Esporas Bacterianas/metabolismo , alfa-Glucosidasas/metabolismoRESUMEN
This study was aimed to investigate the presence of Bacillus coagulans vegetative cells in the intestine and fecal samples in rats fed B. coagulans spores as well as to estimate the ratios of spores and vegetative cells in these samples. A two-step process has been developed to enumerate B. coagulans in different mixed bacterial samples, specifically (1) observation of yellow ring formation on modified GYEA medium upon incubation at 55°C, (2) microscopic examination of spore formation after 7 d of incubation. Our results have demonstrated the presence of vegetative cells in the intestinal and fecal samples in rats fed B. coagulans spores. The ratios of B. coagulans spores and vegetative cells in cecal fluid, colonic content, and feces were approximately 2:8, 2:8, and 4:6, respectively. The existence of B. coagulans vegetative cells improved the intestinal milieu through an elevated short-chain fatty acid concentrations, higher fecal moisture, and lower fecal pH.
Asunto(s)
Bacillus coagulans/fisiología , Heces/microbiología , Intestinos/microbiología , Esporas Bacterianas/aislamiento & purificación , Animales , Ácidos Grasos/metabolismo , Concentración de Iones de Hidrógeno , Intestinos/citología , Masculino , Probióticos , Ratas , Ratas Sprague-DawleyRESUMEN
Mesophilic and thermophilic spore-forming bacteria represent a challenge to the dairy industry, as these bacteria are capable of surviving adverse conditions associated with processing and sanitation and eventually spoil dairy products. The dairy farm environment, including soil, manure, silage, and bedding, has been implicated as a source for spores in raw milk. High levels of spores have previously been isolated from bedding, and different bedding materials have been associated with spore levels in bulk tank (BT) raw milk; however, the effect of different bedding types, bedding management practices, and bedding spore levels on the variance of spore levels in BT raw milk has not been investigated. To this end, farm and bedding management surveys were administered and unused bedding, used bedding, and BT raw milk samples were collected from dairy farms (1 or 2 times per farm) across the United States over 1 yr; the final data set included 182 dairy farms in 18 states. Bedding suspensions and BT raw milk were spore pasteurized (80°C for 12 min), and mesophilic and thermophilic spores were enumerated. Piecewise structural equation modeling analysis was used to determine direct and indirect pathways of association among farm and bedding practices, levels of spores in unused and used bedding, and levels of spores in BT raw milk. Separate models were constructed for mesophilic and thermophilic spore levels. The analyses showed that bedding material had a direct influence on levels of spores in unused and used bedding as well as an indirect association with spore levels in BT raw milk through used bedding spore levels. Specific bedding and farm management practices as well as cow hygiene in the housing area were associated with mesophilic and thermophilic spore levels in unused bedding, used bedding, and BT raw milk. Notably, levels of spores in used bedding were positively related to those in unused bedding, and used bedding spore levels were positively related to those in BT raw milk. The results of this study increase the understanding of the levels and ecology of mesophilic and thermophilic spores in raw milk, emphasize the possible role of bedding as a source of spores on-farm, and present opportunities for dairy producers to reduce spore levels in BT raw milk.