High-throughput screening for aroma production in food fermentations.

A microtiter plate (MTP) method was developed to screen 1064 unique microorganisms-substrate fermentations for production of 68 target aroma compounds. Based on the number of hits identified by GC-MS, 50 fermentations were repeated at 50-mL scale in flasks. Comparison of GC-MS data showed that scaling up from MTP to flask did not generally result in large differences between the volatile profiles, even with a wide variety of substrates (juice, food slurry and food side-streams) and microorganisms (yeast, bacteria and fungi) used. From the screening results, Lactobacillus plantarum fermentation of chilli pepper was further studied as a high amount of phenols, especially guaiacol and 4-ethylphenol, was produced after fermentation. From HPLC-MS and sensory analysis, capsaicin was shown to be a probable precursor for these phenols and a potential mechanism was proposed. The protocol described herein to screen aroma compounds from fermentation of agri-food products and side streams can support development of clean label flavourful food ingredients.

Efficient gene targeting in Penicillium chrysogenum using novel Agrobacterium-mediated transformation approaches.

The industrial production of ß-lactam antibiotics by Penicillium chrysogenum has increased tremendously over the last decades, however, further optimization via classical strain and process improvement has reached its limits. The availability of the genome sequence provides new opportunities for directed strain improvement, but this requires the establishment of an efficient gene targeting (GT) system. Recently, mutations affecting the non-homologous end joining (NHEJ) pathway were shown to increase GT efficiencies following PEG-mediated DNA transfer in P. chrysogenum from 1% to 50%. Apart from direct DNA transfer many fungi can efficiently be transformed using the T-DNA transfer system of the soil bacterium Agrobacterium tumefaciens, however, for P. chrysogenum no robust system for Agrobacterium-mediated transformation was available. We obtained efficient AMT of P. chrysogenum spores with the nourseothricin acetyltransferase gene as selection marker, and using this system we investigated if AMT in a NHEJ mutant background could further enhance GT efficiencies. In general, AMT resulted in higher GT efficiencies than direct DNA transfer, although the final frequencies depended on the Agrobacterium strain and plasmid backbone used. Providing overlapping and complementing fragments on two different plasmid backbones via the same Agrobacterium host was shown to be most effective. This so-called split-marker or bi-partite method resulted in highly efficient GT (>97%) almost exclusively without additional ectopic T-DNA insertions. As this method provides for an efficient GT method independent of protoplasts, it can be applied to other fungi for which no protoplasts can be generated or for which protoplast transformation leads to varying results.

Abiotic and microbiotic factors controlling biofilm formation by thermophilic sporeformers.

One of the major concerns in the production of dairy concentrates is the risk of contamination by heat-resistant spores from thermophilic bacteria. In order to acquire more insight in the composition of microbial communities occurring in the dairy concentrate industry, a bar-coded 16S amplicon sequencing analysis was carried out on milk, final products, and fouling samples taken from dairy concentrate production lines. The analysis of these samples revealed the presence of DNA from a broad range of bacterial taxa, including a majority of mesophiles and a minority of (thermophilic) spore-forming bacteria. Enrichments of fouling samples at 55°C showed the accumulation of predominantly Brevibacillus and Bacillus, whereas enrichments at 65°C led to the accumulation of Anoxybacillus and Geobacillus species. Bacterial population analysis of biofilms grown using fouling samples as an inoculum indicated that both Anoxybacillus and Geobacillus preferentially form biofilms on surfaces at air-liquid interfaces rather than on submerged surfaces. Three of the most potent biofilm-forming strains isolated from the dairy factory industrial samples, including Geobacillus thermoglucosidans, Geobacillus stearothermophilus, and Anoxybacillus flavithermus, have been characterized in detail with respect to their growth conditions and spore resistance. Strikingly, Geobacillus thermoglucosidans, which forms the most thermostable spores of these three species, is not able to grow in dairy intermediates as a pure culture but appears to be dependent for growth on other spoilage organisms present, probably as a result of their proteolytic activity. These results underscore the importance of abiotic and microbiotic factors in niche colonization in dairy factories, where the presence of thermophilic sporeformers can affect the quality of end products.

2'-Fucosyllactose inhibits proliferation of Clostridioides difficile ATCC 43599 in the CDi-screen, an in vitro model simulating Clostridioides difficile infection.

Background: Clostridioides difficile is a Gram-positive anaerobic bacterium that can produce the toxins TcdA and/or TcdB and is considered an opportunistic pathogen. C. difficile is mainly transmitted as endospores, which germinate to produce the pathogenic vegetative cells under suitable conditions in the gut. To efficiently screen novel therapeutic- interventions against the proliferation of C. difficile within a complex microbial community, platforms are needed that facilitate parallel experimentation. In order to allow for screening of novel interventions a medium-to-high throughput in vitro system is desirable. To this end, we have developed the 96-well CDi-screen platform that employs an adapted simulated ileal effluent medium (CDi-SIEM) and allows for culturing of pathogenic C. difficile. Methods: C. difficile strain ATCC 43599 was inoculated in the form of vegetative cells and spores into the CDi-screen in the presence and absence of a cultured fecal microbiota and incubated for 48h. To demonstrate its utility, we investigated the effect of the human milk oligosaccharide 2'-Fucosyllactose (2'-FL) at 4 and 8 mg/mL on C. difficile outgrowth and toxin production in the CDi-screen. The test conditions were sampled after 24 and 48 hours. C. difficile -specific primers were used to monitor C. difficile growth via qPCR and barcoded 16S rRNA gene amplicon sequencing facilitated the in-depth analysis of gut microbial community dynamics. Results: C. difficile ATCC 43599 proliferated in CDi-SIEM, both when inoculated as spores and as vegetative cells. The strain reached cell numbers expressed as C. difficile genome equivalents of up to 10 8 cells per mL after 24h of incubation. 2'-FL significantly inhibited the outgrowth of the ATTC 43599 strain within a complex human gut microbial community in the CDi-screen. In addition, a dose-dependent modulation of the gut microbial community composition by 2'-FL supplementation was detected, with a significant increase in the relative abundance of the genus Blautia in the presence of 2'-FL. Conclusion: The CDi-screen is suitable for studying C. difficile proliferation in a complex gut ecosystem and for screening for anti-pathogenic interventions that target C. difficile directly and/or indirectly through interactions with the gut microbiota. Different doses of compounds such as in this study the dose of the human milk oligosaccharide 2'-FL can be screened for efficacy in the inhibition of C. difficile proliferation.

Highly efficient gene targeting in Penicillium chrysogenum using the bi-partite approach in deltalig4 or deltaku70 mutants.

Inactivating the non-homologous end-joining (NHEJ) pathway is a well established method to increase gene targeting (GT) efficiencies in filamentous fungi. In this study we have compared the effect of inactivating the NHEJ genes ku70 or lig4 on GT in the industrial penicillin producer Penicillium chrysogenum. Deletion of both genes resulted in strongly increased GT efficiencies at three different loci but not higher than 70%, implying that other, yet uncharacterized, recombination pathways are still active causing a part of the DNA to be integrated via non-homologous recombination. To further increase the GT efficiency we applied the bi-partite approach, in which the DNA fragment for integration was split in two non-functional overlapping parts that via homologous recombination invivo can form a functional selection marker. The combined NHEJ mutant and bi-partite approach further increased GT frequencies up to approximately 90%, which will enable the efficient high throughput engineering of the P. chrysogenum genome. We expect that this combined approach will function with similar high efficiencies in other filamentous fungi.

Methods to assess the effect of meat processing on viability of Toxoplasma gondii: towards replacement of mouse bioassay by in vitro testing.

Consumption of meat containing viable tissue cysts is considered one of the main sources of human infection with Toxoplasma gondii. In contrast to fresh meat, raw meat products usually undergo processing, including salting and mixing with other additives such as sodium acetate and sodium lactate, which affects the viability of T. gondii. However, the experiments described in the literature are not always performed in line with the current processing methods applied in industry. It was our goal to study the effect of salting and additives according to the recipes used by industrial producers. Mouse or cat bioassay is the 'gold standard' to demonstrate the presence of viable T. gondii. However, it is costly, time consuming and for ethical reasons not preferred for large-scale studies.Therefore, we first aimed to develop an alternative for mouse bioassay that can be used to determine the effect of processing on the viability of T. gondii tissue cysts. The assays studied were (i) a cell culture method to determine the parasite's ability to multiply, and (ii) a propidium monoazide (PMA) dye-based assay to selectively detect DNA from intact parasites. Processing experiments were performed with minced meat incubated for 20 h with low concentrations of NaCl, sodium lactate and sodium acetate. NaCl appeared to be the most effective ingredient with only one or two out of eight mice infected after inoculation with pepsin-digest of portions processed with 1.0, 1.2 and 1.6% NaCl. Results of preliminary experiments with the PMA-based method were inconsistent and did not sufficiently discriminate between live and dead parasites. In contrast, the cell culture method showed promising results, but further optimization is needed before it can replace or reduce the number of mouse bioassays needed. In future, standardised in vitro methods are necessary to allow more extensive testing of product-specific processing methods, thereby providing a better indication of the risk of T. gondii infection for consumers.

Functional characterization of excision repair and RecA-dependent recombinational DNA repair in Campylobacter jejuni.

The presence and functionality of DNA repair mechanisms in Campylobacter jejuni are largely unknown. In silico analysis of the complete translated genome of C. jejuni NCTC 11168 suggests the presence of genes involved in methyl-directed mismatch repair (MMR), nucleotide excision repair, base excision repair (BER), and recombinational repair. To assess the functionality of these putative repair mechanisms in C. jejuni, mutS, uvrB, ung, and recA knockout mutants were constructed and analyzed for their ability to repair spontaneous point mutations, UV irradiation-induced DNA damage, and nicked DNA. Inactivation of the different putative DNA repair genes did not alter the spontaneous mutation frequency. Disruption of the UvrB and RecA orthologues, but not the putative MutS or Ung proteins, resulted in a significant reduction in viability after exposure to UV irradiation. Assays performed with uracil-containing plasmid DNA showed that the putative uracil-DNA glycosylase (Ung) protein, important for initiation of the BER pathway, is also functional in C. jejuni. Inactivation of recA also resulted in a loss of natural transformation. Overall, the data indicate that C. jejuni has multiple functional DNA repair systems that may protect against DNA damage and limit the generation of genetic diversity. On the other hand, the apparent absence of a functional MMR pathway may enhance the frequency of on-and-off switching of phase variable genes typical for C. jejuni and may contribute to the genetic heterogeneity of the C. jejuni population.

Successional Dynamics in the Gut Microbiome Determine the Success of Clostridium difficile Infection in Adult Pig Models.

Clostridium difficile infections (CDI) are a major cause of antibiotic-associated diarrhea. It is hypothesized that CDI develops due to the antibiotic-induced disruption of the intestinal microbial community structure, which allows C. difficile to flourish. Here, we pre-treated weaned pigs with the antibiotics Clindamycin or Ciprofloxacin for 1 day, and subsequently inoculated them with a human and pig enteropathogenic C. difficile strain 078 spores. Body temperature, clinical signs of disease, and the fecal microbiome were monitored daily for 15 days. Clindamycin had a stronger effect on the pigs than Ciprofloxacin, resulting in drastic shifts in the fecal microbiome, decreases in microbial diversity and significant increases in body temperature, even in the absence of C. difficile. Fecal shedding of C. difficile was detectable for 3 and 9 days in Ciprofloxacin and Clindamycin treated pigs inoculated with C. difficile, respectively, and in both cases decreased cell proliferation rates were detected in colon tissue. The timing of C. difficile shedding coincided with the decrease in a large cluster of Firmicutes following Clindamycin treatment, a pattern which was also independent of C. difficile inoculation. The observed community patterns suggest that successional dynamics following antibiotic treatment facilitate C. difficile establishment. The similarities between the microbiome responses observed in our study and those previously reported in CDI-infected humans further support the utility of adult pigs as models for the study of CDI.

Amplicon sequencing for the quantification of spoilage microbiota in complex foods including bacterial spores.

BACKGROUND: Spoilage of food products is frequently caused by bacterial spores and lactic acid bacteria. Identification of these organisms by classic cultivation methods is limited by their ability to form colonies on nutrient agar plates. In this study, we adapted and optimized 16S rRNA amplicon sequencing for quantification of bacterial spores in a canned food matrix and for monitoring the outgrowth of spoilage microbiota in a ready-to-eat food matrix. RESULTS: The detection limit of bar-coded 16S rRNA amplicon sequencing was determined for the number of bacterial spores in a canned food matrix. Analysis of samples from a canned food matrix spiked with a mixture of equinumerous spores from the thermophiles, Geobacillus stearothermophilus and Geobacillus thermoglucosidans, and the mesophiles, Bacillus sporothermodurans, Bacillus cereus, and Bacillus subtilis, led to the detection of these spores with an average limit of 2 × 10(2) spores ml(-1). The data were normalized by setting the number of sequences resulting from DNA of an inactivated bacterial species, present in the matrix at the same concentration in all samples, to a fixed value for quantitative sample-to-sample comparisons. The 16S rRNA amplicon sequencing method was also employed to monitor population dynamics in a ready-to-eat rice meal, incubated over a period of 12 days at 7 °C. The most predominant outgrowth was observed by the genera Leuconostoc, Bacillus, and Paenibacillus. Analysis of meals pre-treated with weak acids showed inhibition of outgrowth of these three genera. The specificity of the amplicon synthesis was improved by the design of oligonucleotides that minimize the amplification of 16S rRNA genes from chloroplasts originating from plant-based material present in the food. CONCLUSION: This study shows that the composition of complex spoilage populations, including bacterial spores, can be monitored in complex food matrices by bar-coded amplicon sequencing in a quantitative manner. In order to allow sample-to-sample comparisons, normalizations based on background DNA are described. This method offers a solution for the identification and quantification of spoilage microbiota, which cannot be cultivated under standard laboratory conditions. The study indicates variable detection limits among species of bacterial spores resulting from differences in DNA extraction efficiencies.

Rrp5p, a trans-acting factor in yeast ribosome biogenesis, is an RNA-binding protein with a pronounced preference for U-rich sequences.

Rrp5p is a trans-acting factor important for biogenesis of both the 40S and 60S subunit of the Saccharomyces cerevisiae ribosome. The protein contains 12 tandemly repeated S1 RNA binding motifs in its N-terminal region, suggesting the ability to interact directly with the pre-rRNA. In vitro binding studies, using immunopurified Rrp5p and in vitro transcribed, 32P-UTP-labeled RNA fragments, revealed that Rrp5p is a general RNA-binding protein with a strong preference for single-stranded sequences rich in uridines. Co-immunoprecipitation studies in yeast cells expressing ProtA-tagged Rrp5p showed that the protein is still associated with pre-ribosomal particles containing 27SA2 pre-rRNA but not with particles containing the 27SB precursor. Thus, Rrp5p appears to dissociate from the 66S pre-ribosome upon or immediately after further processing of 27SA2 pre-rRNA, suggesting the presence of (an) important binding site(s) within the 3'-terminal portion of ITS1. The location of these possible binding site(s) was further delimited using rrp2-1 mutant cells, which accumulate the 5'-extended 5.8S pre-rRNA species. The results indicate that association of Rrp5p with the pre-ribosome is abolished upon removal of a 30-nt region downstream from site A2, which contains two short, single-stranded U stretches. Sequence comparison shows that only the most 5' of these two U-rich stretches is conserved among yeast species whose ITS1 can functionally replace the S. cerevisiae spacer. The implications for the role of Rrp5p in yeast ribosome biogenesis are discussed.

Xanthobacter flavus employs a single triosephosphate isomerase for heterotrophic and autotrophic metabolism.

The expression of the cbb and gap-pgk operons of Xanthobacter flavus encoding enzymes of the Calvin cycle is regulated by the transcriptional regulator CbbR. In order to identify other genes involved in the regulation of these operons, a mutant was isolated with a lowered activity of a fusion between the promoter of the cbb operon and the reporter gene lacZ. This mutant was unable to grow autotrophically and had a reduced growth rate on medium supplemented with gluconate or succinate. The regulation of the gap-pgk operon in the mutant was indistinguishable from the wild-type strain, but induction of the cbb operon upon transition to autotrophic growth conditions was delayed. Complementation of the mutant with a genomic library of X. flavus resulted in the isolation of a 1.1 kb ApaI fragment which restored autotrophic growth of the mutant. One open reading frame (ORF) was present on the ApaI fragment, which could encode a protein highly similar to triosephosphate isomerase proteins from other bacteria. Cell extracts of the mutant grown under glycolytic or gluconeogenic conditions had severely reduced triosephosphate isomerase activities. The ORF was therefore identified as tpi, encoding triosephosphate isomerase. The tpi gene is not linked to the previously identified operons encoding Calvin cycle enzymes and therefore represents a third transcriptional unit required for autotrophic metabolism.

Generation of Campylobacter jejuni genetic diversity in vivo.

Molecular epidemiology studies suggest that horizontal genetic exchange is a major cause of pathogen biodiversity. We tested this concept for the bacterial enteropathogen Campylobacter jejuni by seeking direct in vivo evidence for the exchange of genetic material among Campylobacter strains. For this purpose, two antibiotic resistance markers were inserted into the hipO or htrA gene of genetically distinct and naturally transformable C. jejuni strains. Genetic exchange of the resistance markers was analysed after co-cultivation of homologous and heterologous strains in vitro and in vivo during experimental infection of chickens. Double-resistant recombinants were obtained both in vitro and from the chicken intestine for all combinations of strains tested. Bidirectional genetic exchange of DNA between homologous and heterologous strains was confirmed by Southern blotting in combination with flaA polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), amplified fragment length polymorphism (AFLP) and pulsed field gel electrophoresis (PFGE). Extensive PFGE analyses of isolated recombinants indicated the frequent occurrence of genetic rearrangements during the experimental infection, in addition to the homologous recombination of the antibiotic resistance genes. Together, the data indicate unequivocally that interstrain genetic exchange as well as intragenomic alterations do occur in vivo during C. jejuni infection. These events probably explain the genome plasticity observed for this pathogen.