The Digestibility of Hibiscus sabdariffa L. Polyphenols Using an In Vitro Human Digestion Model and Evaluation of Their Antimicrobial Activity

Hibiscus sabdariffa L. (H.s.) is a polyphenolic-rich plant commonly consumed either as a beverage or spice. The aim of the present study was to evaluate the in vitro digestibility of H.s. polyphenols using an in vitro model of digestion which simulates the human stomach and small intestine. The bioaccessible polyphenols released in the digested samples were analyzed by liquid chromatography coupled to photodiode array and mass spectrometry detection. H.s. anthocyanins (cyanidin-3-O-sambubioside and delphinidin-3-O-sambubioside) content drastically dropped during the digestion process from 2.91 ± 0.03 µg g−1 and 8.53 ± 0.08 µg g−1 (w/w) CG (Cyanidin-glucoside) in the raw extract, respectively, to 0.12 ± 0.01 µg g−1 0.12 ± 0.01 µg g−1 (w/w) CG at the end of duodenal digestion. Total polyphenols also have shown a decrease from 1192.65 ± 30.37 µg g−1 (w/w) in the raw extract to 282.24 ± 7.21 µg g−1 (w/w) by the end of gastric digestion, in contrast to their increase by the end of duodenal digestion 372.91 ± 3.97 µg g−1 (w/w). On the other hand, the decrease in certain compounds (e.g., caffeoylquinicandcoumaroylquinic acids) was observed during gastric digestion resulting in an increase of quinic acid in the duodenal aliquots, thus suggesting that this compound was derived from the degradation of the more complex hydroxycinnamic acids. H.s. extract also exhibited a bacteriostatic effect against Staphylococcus aureus ATCC 6538 (MIC of 2.5 mg mL−1) and a bactericidal effect against a food isolate of Listeria monocytogenes (MBC of 2.5 mg mL−1). The undigested polyphenols of H.s. in the upper gastrointestinal tract enters the colon, where they are metabolized by the gut microbiota. The present study results showed that resistance of H.s. polyphenols during gastrointestinal digestion might affect their uptake, resulting in a decrease in their digestibility.

Application of a strain-level shotgun metagenomics approach on food samples: resolution of the source of a Salmonella food-borne outbreak

Food-borne outbreak investigation currently relies on the time-consuming and challenging bacterial isolation from food, to be able to link food-derived strains to more easily obtained isolates from infected people. When no food isolate can be obtained, the source of the outbreak cannot be unambiguously determined. Shotgun metagenomics approaches applied to the food samples could circumvent this need for isolation from the suspected source, but require downstream strain-level data analysis to be able to accurately link to the human isolate. Until now, this approach has not yet been applied outside research settings to analyse real food-borne outbreak samples. In September 2019, a Salmonella outbreak occurred in a hotel school in Bruges, Belgium, affecting over 200 students and teachers. Following standard procedures, the Belgian National Reference Center for human salmonellosis and the National Reference Laboratory for Salmonella in food and feed used conventional analysis based on isolation, serotyping and MLVA (multilocus variable number tandem repeat analysis) comparison, followed by whole-genome sequencing, to confirm the source of the contamination over 2 weeks after receipt of the sample, which was freshly prepared tartar sauce in a meal cooked at the school. Our team used this outbreak as a case study to deliver a proof of concept for a short-read strain-level shotgun metagenomics approach for source tracking. We received two suspect food samples: the full meal and some freshly made tartar sauce served with this meal, requiring the use of raw eggs. After analysis, we could prove, without isolation, that Salmonella was present in both samples, and we obtained an inferred genome of a Salmonella enterica subsp. enterica serovar Enteritidis that could be linked back to the human isolates of the outbreak in a phylogenetic tree. These metagenomics-derived outbreak strains were separated from sporadic cases as well as from another outbreak circulating in Europe at the same time period. This is, to our knowledge, the first Salmonella food-borne outbreak investigation uniquely linking the food source using a metagenomics approach and this in a fast time frame.

Molecular Physiological Characterization of a High Heat Resistant Spore Forming Bacillus subtilis Food Isolate

Bacterial endospores (spores) are among the most resistant living forms on earth. Spores of Bacillus subtilis A163 show extremely high resistance to wet heat compared to spores of laboratory strains. In this study, we found that spores of B. subtilis A163 were indeed very wet heat resistant and released dipicolinic acid (DPA) very slowly during heat treatment. We also determined the proteome of vegetative cells and spores of B. subtilis A163 and the differences in these proteomes from those of the laboratory strain PY79, spores of which are much less heat resistant. This proteomic characterization identified 2011 proteins in spores and 1901 proteins in vegetative cells of B. subtilis A163. Surprisingly, spore morphogenic protein SpoVM had no homologs in B. subtilis A163. Comparing protein expression between these two strains uncovered 108 proteins that were differentially present in spores and 93 proteins differentially present in cells. In addition, five of the seven proteins on an operon in strain A163, which is thought to be primarily responsible for this strain’s spores high heat resistance, were also identified. These findings reveal proteomic differences of the two strains exhibiting different resistance to heat and form a basis for further mechanistic analysis of the high heat resistance of B. subtilis A163 spores.

Antibacterial properties of Tualang, Acacia and Yemeni Sumur honey against selected food spoilage bacteria and foodborne pathogens

The antibacterial activity of honey is mainly credited to its acidity, osmolarity and enzymatic generation of hydrogen peroxide via glucose oxidase. Additional honey components, such as aromatic acids or phenolic compounds, also contribute to the overall antibacterial activity. The level of antibacterial activities found in honey varies with different types of honey, due to mainly the composition, percentage as well as the nature of the sugars present in the honey. This study aimed to evaluate the antibacterial activity of four types of honey, namely Tualang honey (TH1), Tualang honey (TH2), Acacia honey (AH) and Yemeni Sumur honey (YSH). Nine bacterial strains were used. Disc diffusion, well diffusion, minimum inhibitory concentration (MIC), Minimum bactericidal concentration (MBC), and time-kill methods were performed to reveal the antibacterial potential of the selected honey. The MIC values ranged between 12.5 to 50% for both TH1 and YSH while for TH2, and AH it ranged between 25 to 50%. For MBC, it ranged from 25 to 50%. The time-kill in TH1 Staphylococcus aureus (food isolate) showed total inhibition at 6 hrs in 2 X MIC, and for Staphylococcus aureus ATCC 29737 was 3.84 log CFU/g at the 6 hrs. Physicochemical quality of honey resulted as follows: the pH of the honey samples was acidic in nature ranging between 3.69 to 3.94, and the aw of the honey samples were between 0.53 to 0.69. For colour analysis, YSH was observed to has the maximum lightness and yellowness, and TH1 has the maximum redness. While, AH had a minimum lightness, redness, and yellowness.

ASSESSMENT OF PROBIOTIC PROPERTIES OF THE STRAIN LACTOBACILLUS PARACASEI D6 AND ITS ANTICANDIDAL ACTIVITIES

Objective: The objective of the study was to determine the probiotic properties of a strain Lactobacillus paracasei D6 (GenBank Accession No. KJ867173) and to assess its antifungal activities against certain Candida strains. Methods: Antifungal activities of Lactobacillus paracasei D6 was tested against Candida tropicalis BSS7 and C. albicans MTCC 3017. The cell-free extract was tested against the formation of biofilm and germ tube of the aforementioned Candida strains. Different probiotic activities such as tolerance to simulated gastrointestinal fluid, adhesion to hydrocarbons, and Caco-2 cell line were evaluated for the strain Lactobacillus paracasei D6. Inhibition of Candida strains to Caco-2 cell line was also tested. The strains were identified using gene sequencing followed by phylogenetic tree construction. Results: The probiotic properties of Lactobacillus paracasei D6 were found to be statistically comparable with a standard Lactobacillus plantarum MTCC 1407. The biofilm and germ tube formation patterns of the food spoilage isolate C. tropicalis BSS7 were found to be similar to the strain C. albicans MTCC 3017. Cell-free extract of Lactobacillus paracasei D6 exhibited minimum biofilm inhibitory concentration of 0.438 mg/ml against C. tropicalis BSS7, which was found to be sufficient to inhibit its germ tube formation. The adhesion of C. tropicalis BSS7 to the epithelial Caco-2 cell line was also significantly reduced by the antifungal metabolites. Conclusion: This work gives insight on possible virulent nature of the food isolate C. tropicalis BSS7. Exometabolites produced by L. paracasei D6 were able to inhibit growth, biofilm and germ tube formation of both the strains. With this work, the authors envisage the beginning of an alternative strategy for anticandidal therapy.

Pepper Bacterial Spot Control by Bacillus velezensis: Bioprocess Solution

Pepper bacterial spot is one of the most severe plant diseases in terms of infection persistence and economic losses when it comes to fresh pepper fruits used in nutrition and industrial processing. In this study, Bacillus velezensis IP22 isolated from fresh cheese was used as a biocontrol agent of pepper bacterial spot, whose main causal agent is the cosmopolitan pathogen Xanthomonas euvesicatoria. After optimization of the cultivation medium composition aimed at maximizing of the antimicrobial activity against X. euvesicatoria and validation of the optimized medium at the scale of a laboratory bioreactor, in planta tests were performed. The results have showed significant suppression of bacterial spot symptoms in pepper plants by the produced biocontrol agent, as well as reduction of disease spreading on the healthy (uninoculated) pepper leaves. Furthermore, HPLC-MS (high pressure liquid chromatography–mass spectrometry) analysis was employed to examine antimicrobial metabolites produced by B. velezensis IP22, where lipopeptides were found with similar m/z values compared to lipopeptides from fengycin and locillomycin families. The bioprocess solution developed at the laboratory scale investigated in this study represents a promising strategy for production of pepper bacterial spot biocontrol agent based on B. velezensis IP22, a food isolate with a great perspective for application in plant protection.

A Practical Method to Implement Strain-Level Metagenomics-Based Foodborne Outbreak Investigation and Source Tracking in Routine

The management of a foodborne outbreak depends on the rapid and accurate identification of the responsible food source. Conventional methods based on isolation of the pathogen from the food matrix and target-specific real-time polymerase chain reactions (qPCRs) are used in routine. In recent years, the use of whole genome sequencing (WGS) of bacterial isolates has proven its value to collect relevant information for strain characterization as well as tracing the origin of the contamination by linking the food isolate with the patient’s isolate with high resolution. However, the isolation of a bacterial pathogen from food matrices is often time-consuming and not always successful. Therefore, we aimed to improve outbreak investigation by developing a method that can be implemented in reference laboratories to characterize the pathogen in the food vehicle without its prior isolation and link it back to human cases. We tested and validated a shotgun metagenomics approach by spiking food pathogens in specific food matrices using the Shiga toxin-producing Escherichia coli (STEC) as a case study. Different DNA extraction kits and enrichment procedures were investigated to obtain the most practical workflow. We demonstrated the feasibility of shotgun metagenomics to obtain the same information as in ISO/TS 13136:2012 and WGS of the isolate in parallel by inferring the genome of the contaminant and characterizing it in a shorter timeframe. This was achieved in food samples containing different E. coli strains, including a combination of different STEC strains. For the first time, we also managed to link individual strains from a food product to isolates from human cases, demonstrating the power of shotgun metagenomics for rapid outbreak investigation and source tracking.

Case report: whole genome sequencing based investigation of maternal-neonatal listeriosis in Sichuan, China

Background Neonatal listeriosis is a rare but severe disease manifesting as septicemia and central nervous system (CNS) infections with a high fatality rate of around 20 to 30%. Whole genome sequencing (WGS) is a promising technique for pathogen identification and infection source tracing with its high resolution. Case presentation A case of neonatal sepsis with listeriosis was reported with positive blood culture for Listeria monocytogenes. The case was investigated to confirm the vertical transmission of the infection and identify the potential food source of the maternal L. monocytogenes infection using WGS. L. monocytogenes was isolated from the neonate’s blood sample the day after caesarean delivery and from the mother’s genital and pudenda swab samples 5 days and 13 days after caesarean delivery. WGS showed that the isolate from the neonate was identical to the genome type of the isolates from the mother, with only one of the 4 isolates from the mother differing by one single nucleotide polymorphism (SNP). By WGS, one L. monocytogenes isolate from a ready-to-eat (RTE) meat sample in the patients’ community market shared the same sequence type but was ruled out as the cause of infection, with 57 SNP differences to the strain causing the maternal-neonatal infection. The food isolate also carried a novel plasmid pLM1686 that harbored heavy metal resistance genes. After caesarean section, the mother was treated with a third generation cephalosporin which L. monocytogenes is naturally resistant to, which may explain why genital and pudenda swabs were still culture-positive for L. monocytogenes 13 days after delivery. Conclusions Genital swab culture for L. monocytogenes had been informative in the diagnosis of maternal listeriosis in this case. The high resolution of WGS confirmed the maternal-neonatal transmission of L. monocytogenes infection and ruled out the L. monocytogenes contaminated RTE meat from the local market as the direct source of the mother’s infection.

Listeriosis: genotyping as a key for identification a possible source of infection

Objective. To perform a comparative analysis of clinical and food isolates of Listeria monocytogenes collected in the European part of Russia in 2018–2019. Materials and Methods. We used multilocus sequencing (MLST), supplemented by virulence loci, including fragments of internalin genes (MvLST, Multi-virulent-locus sequence typing), followed by phylogenetic analysis. Results. The main diagnoses for clinical isolates were prenatal and neonatal listeriosis and meningitis. Clinical isolates predominantly belonged to phylogenetic line II with the predominance of ST7, which was also the most abundant in food isolates. The second most common occurrence in food isolates was ST121, widely distributed in Europe. Isolates of phylogenetic line I in the group of clinical cultures in three cases were represented by ST6, detected during outbreaks of listeriosis in Europe 2015–2018 and South Africa in 2017–2018. Only in one isolate from food belonged to the phylogenetic lineage I. In general, the diversity of food isolate genotypes was significantly higher than clinical isolates. The analysis of virulence loci revealed a new internalin A allele and a new internalin genes profile (IP) in isolate ST7 from food. Conclusions. L. monocytogenes of the most common ST7 is autochthonous in Russia; cases of listeriosis caused by the ST6 bacterium are most likely imported. Based on the analysis of the diversity of ST and IP of L. monocytogenes identified in Russia, a rapid diagnosis scheme for epidemiological investigation is proposed.