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Background The microbiome of cheese is diverse, even within a variety. The metagenomics of cheese is dependent on a vast array of biotic and abiotic factors. Biotic factors include the population of microbiota and their resulting cellular metabolism. Abiotic factors, including the pH, water activity, fat, salt, and moisture content of the cheese matrix, as well as environmental conditions (temperature, humidity, and location of aging), influence the biotic factors. This study assessed the metagenomics of commercial Gouda cheese prepared using pasteurized or unpasteurized cow milk or pasteurized goat milk via 16S rDNA sequencing. Results Results were analyzed and compared based on milk pasteurization and source, spatial variability (core, outer, and under the rind), and length of aging (2–4 up to 12–18 months). The dominant organisms in the Gouda cheeses, based on percentage of sequence reads identified at the family or genus levels, were Bacillaceae , Lactococcus , Lactobacillus , Streptococcus , and Staphylococcus . More genus- or family-level (e.g. Bacillaceae ) identifications were observed in the Gouda cheeses prepared with unpasteurized cow milk (120) compared with those prepared with pasteurized cow milk (92). When assessing influence of spatial variability on the metagenomics of the cheese, more pronounced differences in bacterial genera were observed in the samples taken under the rind; Brachybacterium , Pseudoalteromonas , Yersinia , Klebsiella , and Weissella were only detected in these samples. Lastly, the aging length of the cheese greatly influenced the number of organisms observed. Twenty-seven additional genus-level identifications were observed in Gouda cheese aged for 12–18 months compared with cheese only aged 2–4 months. Conclusions Collectively, the results of this study are important in determining the typical microbiota associated with Gouda cheese and how the microbiome plays a role in safety and quality.

Botulinum neurotoxins are a varied group of protein toxins that share similar structures and modes of activity. They include at least seven serotypes and over forty subtypes that are produced by seven different clostridial species. These bacterial species are not limited strictly to BoNT-producers as neuro-toxigenic and non-neuro-toxigenic members have been identified within each species. The nomenclature surrounding these toxins and associated bacteria has been evolving as new isolations and discoveries have arisen, resulting in challenges in diagnostic reporting, epidemiology and food safety studies, and in the application of therapeutic products. An understanding of the intricacies regarding the nomenclature of BoNTs and BoNT-producing clostridia is crucial for communication that allows for accurate reporting of information that is pertinent to each situation.

At least 40 toxin subtypes of botulinum neurotoxins (BoNTs), a heterogenous group of bacterial proteins, are produced by seven different clostridial species. A key factor that drives the diversity of neurotoxigenic clostridia is the association of bont gene clusters with various genomic locations including plasmids, phages and the chromosome. Analysis of Clostridium sporogenes BoNT/B1 strain CDC 1632, C. argentinense BoNT/G strain CDC 2741, and Clostridium parabotulinum BoNT/B1 strain DFPST0006 genomes revealed bont gene clusters within plasmid-like sequences within the chromosome or nested in large contigs, with no evidence of extrachromosomal elements. A nucleotide sequence (255,474 bp) identified in CDC 1632 shared 99.5% identity (88% coverage) with bont/B1-containing plasmid pNPD7 of C. sporogenes CDC 67071; CDC 2741 contig AYSO01000020 (1.1 MB) contained a ~140 kb region which shared 99.99% identity (100% coverage) with plasmid pRSJ17_1 of C. argentinense BoNT/G strain 89G; and DFPST0006 contig JACBDK0100002 (573 kb) contained a region that shared 100% identity (99%) coverage with the bont/B1-containing plasmid pCLD of C. parabotulinum Okra. This is the first report of full-length plasmid DNA-carrying complete neurotoxin gene clusters integrated in three distinct neurotoxigenic species: C. parabotulinum, C. sporogenes and C. argentinense.

The microbial safety concern associated with thermally processed extended shelf life (ESL) refrigerated foods is based on adequate elimination of spore-forming pathogens such as nonproteolytic Clostridium botulinum types B, E, and F. These pathogens are traditionally regarded as targets for validation of thermally processed ESL foods. However, their use for research is restricted due to their designation as select agents. In this study, the thermal resistances of spores of 10 nonproteolytic C. botulinum types B and F and seven psychrotrophic Bacillus cereus strains were evaluated in ACES (N-(2-acetamido)-2-aminoethanesulfonic acid) buffer (0.05 M, pH 7.00) and compared to determine whether any of the B. cereus strains could serve as a nonselect agent for establishing thermal processes for ESL refrigerated foods. Thermal decimal reduction times (DT-values) of both nonproteolytic C. botulinum types B and F and psychrotrophic B. cereus strains decreased as process temperature increased from 80 to 91°C, and the highest values were obtained at 80°C. All psychrotrophic B. cereus strains tested were more thermally resistant than nonproteolytic C. botulinum types B and F. DT-values of nonproteolytic C. botulinum types B and F decreased to <1.0 min at 87°C, whereas all psychrotrophic B. cereus strains had higher DT-values (i.e., 52.35 to 133.69 min) at the same temperature. Among all psychrotrophic B. cereus strains tested, BC-6A16 had the highest DT-values at any given temperature. The DT-values indicated that the psychrotrophic B. cereus strains were more thermally resistant than the nonproteolytic C. botulinum strains and therefore may be potential target pathogens for thermal process validation of ESL refrigerated foods. However, further comparative challenge studies are needed with a model food system or an ESL refrigerated food to confirm these results.

Cheeses made with unpasteurized milk are a safety concern due to possible contamination with foodborne pathogens. Listeria monocytogenes and Escherichia coli O157:H7 have been implicated in several outbreaks and recalls linked to Gouda cheese made with unpasteurized milk. The U.S. Food and Drug Administration Code of Federal Regulations requires cheeses made with unpasteurized milk to be aged at a minimum of 1.7°C for at least 60 days before entering interstate commerce. The goal of this study was (i) to assess the population dynamics of L. monocytogenes and E. coli O157:H7 during aging of Gouda cheese when the pathogens were inoculated into the unpasteurized milk used for manufacture and (ii) to compare the native microbial populations throughout manufacture and aging. Unpasteurized milk was inoculated with L. monocytogenes at 1 or 3 log CFU/mL or with E. coli O157:H7 at 1 log CFU/mL, and Gouda cheese was manufactured in laboratory-scale or pilot plant-scale settings. Cheeses were stored at 10°C for at least 90 days, and some cheeses were stored up to 163 days. Initial native microflora populations in unpasteurized milk did not differ significantly for laboratory-scale or pilot plant-scale trials, and population dynamics trended similarly throughout cheese manufacture and aging. During manufacture, approximately 81% of the total L. monocytogenes and E. coli O157:H7 populations was found in the curd samples. At an inoculation level of 1 log CFU/mL, L. monocytogenes survived in the cheese beyond 60 days in four of five trials. In contrast, E. coli O157:H7 was detected beyond 60 days in only one trial. At the higher 3-log inoculation level, the population of L. monocytogenes increased significantly from 3.96 ± 0.07 log CFU/g at the beginning of aging to 6.00 ± 0.73 log CFU/g after 150 days, corresponding to a growth rate of 0.04 ± 0.02 log CFU/g/day. The types of native microflora assessed included Enterobacteriaceae, lactic acid bacteria, mesophilic bacteria, and yeasts and molds. Generally, lactic acid and mesophilic bacterial populations remained consistent at approximately 8 to 9 log CFU/g during aging, whereas yeast and mold populations steadily increased. The data from this study will contribute to knowledge about survival of these pathogens during Gouda cheese production and will help researchers assess the risks of illness from consumption of Gouda cheese made with unpasteurized milk.

The impact of high pressure processing on the inactivation of spores of nonproteolytic Clostridium botulinum is important in extended shelf life chilled low-acid foods. The three most resistant C. botulinum strains (Ham-B, Kap 9-B, and 610-F) were selected for comparison of their thermal and pressure-assisted thermal resistance after screening 17 nonproteolytic C. botulinum strains (8 type B, 7 type E, and 2 type F). Spores of strains Ham-B, Kap 9-B, and 610-F were prepared using a biphasic media method, diluted in N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) buffer (0.05 M, pH 7.00) to 10 to 10 CFU/mL, placed into a modified sterile transfer pipette, heat sealed, and subjected to a combination of high pressures (600 to 750 MPa) and high temperatures (80 to 91°C) using laboratory and pilot-scale pressure test systems. Diluted spores from the same crops were placed in nuclear magnetic resonance tubes, which were heat sealed, and subjected to 80 to 91°C in a Fluke 7321 high precision bath with Duratheram S oil as the heat transfer fluid. After incubation for 3 months, survivors in both studies were determined by the five-tube most-probable-number method using Trypticase-peptone-glucose-yeast extract broth. The highest (>5.0) log reductions in spore counts for Ham-B, Kap 9-B, and 610-F occurred at the highest temperature and pressure combination tested (91°C and 750 MPa). Thermal D-values of Ham-B, Kap 9-B, and 610-F decreased as the process temperature increased from 80 to 87°C, decreasing to <1.0 min at 87°C for these strains. Pressure-assisted thermal D-values of Ham-B, Kap 9-B, and 610-F decreased as the process temperature increased from 80 to 91°C with any pressure combination and decreased to <1.0 min as the pressure increased from 600 to 750 MPa at 91°C. Based on the pressure-assisted thermal D-values, pressure exerted a more protective effect on spores of Ham-B, Kap 9-B, and 610-F when processed at 83 to 91°C combined with pressures of 600 to 700 MPa when compared with thermal treatment only. No protective effect was observed when the spores of Ham-B, Kap9-B, and 610-F were treated at lower temperatures (80 to 83°C) in combination with 750 MPa. However, at higher temperatures (87 to 91°C) in combination with 750 MPa, a protective effect was seen for Ham-B, Kap9-B, and 610-F spores based on the calculated pressure-assisted thermal D-values.

The purpose of this study was to determine the inactivation kinetics of the spores of the most resistant proteolytic Clostridium botulinum strains (Giorgio-A and 69-A, as determined from an earlier screening study) and of Clostridium sporogenes PA3679 and to compare the thermal and pressure-assisted thermal resistance of these spores. Spores of these strains were prepared using a biphasic medium method. C. sporogenes PA3679 spores were heat treated before spore preparation. Using laboratory-scale and pilot-scale pressure test systems, spores of Giorgio-A, 69-A, and PA3679 suspended in ACES [N-(2-acetamido)-2-aminoethanesulfonic acid] buffer (pH 7.0) were exposed to various combinations of temperature (93 to 121°C) and pressure (0.1 to 750 MPa) to determine their resistance. More than a 5-log reduction occurred after 3 min at 113°C for spores of Giorgio-A and 69-A and after 5 min at 117°C for spores of PA3679. A combination of high temperatures (93 to 121°C) and pressures yielded greater log reductions of spores of Giorgio-A, 69-A, and PA3679 compared with reduction obtained with high temperatures alone. No survivors from initial levels (>5.0 log CFU) of Giorgio-A and 69-A were detected when processed at a combination of high temperature (117 and 121°C) and high pressure (600 and 750 MPa) for <1 min in a pilot-scale pressure test system. Increasing pressure from 600 to 750 MPa at 117°C decreased the time from 2.7 to 1 min for a >4.5-log reduction of PA3679 spores. Thermal D-values of Giorgio-A, 69-A, and PA3679 spores decreased (i.e., 29.1 to 0.33 min for Giorgio-A, 40.5 to 0.27 min for 69-A, and 335.2 to 2.16 min for PA3679) as the temperature increased from 97 to 117°C. Pressure-assisted thermal D-values of Giorgio-A, 69-A, and PA3679 also decreased as temperature increased from 97 to 121°C at both pressures (600 and 750 MPa) (i.e., 17.19 to 0.15 min for Giorgio-A, 9.58 to 0.15 min for 69-A, and 12.93 to 0.33 min for PA3679 at 600 MPa). At higher temperatures (117 or 121°C), increasing pressure from 600 to 750 MPa had an effect on pressure-assisted thermal D-values of PA3679 (i.e., at 117°C, pressure-assisted thermal D-value decreased from 0.55 to 0.28 min as pressure increased from 600 to 750 MPa), but pressure had no effect on pressure-assisted thermal D-values of Giorgio-A and 69-A. When compared with Giorgio-A and 69-A, PA3679 had higher thermal and pressure-assisted thermal D-values. C. sporogenes PA3679 spores were generally more resistant to combinations of high pressure and high temperature than were the spores of the C. botulinum strains tested in this study.

The brain is organized into networks at multiple resolutions, or scales, yet studies of functional network development typically focus on a single scale. Here, we derive personalized functional networks across 29 scales in a large sample of youths (n = 693, ages 8–23 years) to identify multi-scale patterns of network re-organization related to neurocognitive development. We found that developmental shifts in inter-network coupling reflect and strengthen a functional hierarchy of cortical organization. Furthermore, we observed that scale-dependent effects were present in lower-order, unimodal networks, but not higher-order, transmodal networks. Finally, we found that network maturation had clear behavioral relevance: the development of coupling in unimodal and transmodal networks are dissociably related to the emergence of executive function. These results suggest that the development of functional brain networks align with and refine a hierarchy linked to cognition. Studies of brain network development typically focus on a single scale. Here, the authors derived personalized functional networks across scales, and find that network development systematically adheres to and strengthens hierarchical cortical organization.

The brain is organized into networks at multiple resolutions, or scales, yet studies of functional network development typically focus on a single scale. Here, we derived personalized functional networks across 29 scales in a large sample of youths (n=693, ages 8-23 years) to identify multi-scale patterns of network re-organization related to neurocognitive development. We found that developmental shifts in inter-network coupling systematically adhered to and strengthened a functional hierarchy of cortical organization. Furthermore, we observed that scale-dependent effects were present in lower-order, unimodal networks, but not higher-order, transmodal networks. Finally, we found that network maturation had clear behavioral relevance: the development of coupling in unimodal and transmodal networks dissociably mediated the emergence of executive function. These results delineate maturation of multi-scale brain networks, which varies according to a functional hierarchy and impacts cognitive development.