Genotypic characterization and safety assessment of lactic acid bacteria from indigenous African fermented food products

BACKGROUND: Indigenous fermented food products play an essential role in the diet of millions of Africans. Lactic acid bacteria (LAB) are among the predominant microbial species in African indigenous fermented food products and are used for different applications in the food and biotechnology industries. Numerous studies have described antimicrobial susceptibility profiles of LAB from different parts of the world. However, there is limited information on antimicrobial resistance profiles of LAB from Africa. The aim of this study was to characterize 33 LAB previously isolated from three different African indigenous fermented food products using (GTG)(5)-based rep-PCR, sequencing of the 16S rRNA gene and species-specific PCR techniques for differentiation of closely related species and further evaluate their antibiotic resistance profiles by the broth microdilution method and their haemolytic activity on sheep blood agar plates as indicators of safety traits among these bacteria. RESULTS: Using molecular biology based methods and selected phenotypic tests such as catalase reaction, CO(2) production from glucose, colonies and cells morphology, the isolates were identified as Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus ghanensis, Lactobacillus plantarum, Lactobacillus salivarius, Leuconostoc pseudomesenteroides, Pediococcus acidilactici, Pediococcus pentosaceus and Weissella confusa. The bacteria were susceptible to ampicillin, chloramphenicol, clindamycin and erythromycin but resistant to vancomycin, kanamycin and streptomycin. Variable sensitivity profiles to tetracycline and gentamicin was observed among the isolates with Lb. plantarum, Lb. salivarius, W. confusa (except strain SK9-5) and Lb. fermentum strains being susceptible to tetracycline whereas Pediococcus strains and Lb. ghanensis strains were resistant. For gentamicin, Leuc. pseudomesenteroides, Lb. ghanensis and Ped. acidilactici strains were resistant to 64 mg/L whereas some W. confusa and Lb. plantarum strains had a MIC value of 16 mg/L and 32 mg/L respectively. No β-haemolytic activity was observed, however, α-haemolytic activity was observed in 27% (9) of the strains comprising Lb. salivarius (6), W. confusa (2) and Lb. delbrueckii (1) isolates. CONCLUSIONS: The resistance to kanamycin and vancomycin is probably an intrinsic feature since similar observations were reported in the literature for LAB. Low prevalence of pathogenicity indicator traits were observed among the isolates especially with the presence of poor haemolytic activities and they could therefore be considered as interesting candidates for selection of starter cultures or probiotics for different applications

Draft whole-genome sequence of <em>Bacillus sonorensis </em>strain L12, a source of nonribosomal lipopeptides

The Bacillus sonorensis L12 draft genome sequence is approximately 4,647,754 bp in size with a G+C content of 45.2%. Over 86% of the genome contains protein-encoding genes, including several gene clusters for de novo biosynthesis of the nonribosomal lipopeptides iturin, bacitracin, and fengycin, which could mean that the strain exhibits antifungal effects

Inhibition of IL-10 Production by Maternal Antibodies against Group B Streptococcus GAPDH Confers Immunity to Offspring by Favoring Neutrophil Recruitment

Group B Streptococcus (GBS) is the leading cause of neonatal pneumonia, septicemia, and meningitis. We have previously shown that in adult mice GBS glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an extracellular virulence factor that induces production of the immunosuppressive cytokine interleukin-10 (IL-10) by the host early upon bacterial infection. Here, we investigate whether immunity to neonatal GBS infection could be achieved through maternal vaccination against bacterial GAPDH. Female BALB/c mice were immunized with rGAPDH and the progeny was infected with a lethal inoculum of GBS strains. Neonatal mice born from mothers immunized with rGAPDH were protected against infection with GBS strains, including the ST-17 highly virulent clone. A similar protective effect was observed in newborns passively immunized with anti-rGAPDH IgG antibodies, or F(ab')2 fragments, indicating that protection achieved with rGAPDH vaccination is independent of opsonophagocytic killing of bacteria. Protection against lethal GBS infection through rGAPDH maternal vaccination was due to neutralization of IL-10 production soon after infection. Consequently, IL-10 deficient (IL-10−/−) mice pups were as resistant to GBS infection as pups born from vaccinated mothers. We observed that protection was correlated with increased neutrophil trafficking to infected organs. Thus, anti-rGAPDH or anti-IL-10R treatment of mice pups before GBS infection resulted in increased neutrophil numbers and lower bacterial load in infected organs, as compared to newborn mice treated with the respective control antibodies. We showed that mothers immunized with rGAPDH produce neutralizing antibodies that are sufficient to decrease IL-10 production and induce neutrophil recruitment into infected tissues in newborn mice. These results uncover a novel mechanism for GBS virulence in a neonatal host that could be neutralized by vaccination or immunotherapy. As GBS GAPDH is a structurally conserved enzyme that is metabolically essential for bacterial growth in media containing glucose as the sole carbon source (i.e., the blood), this protein constitutes a powerful candidate for the development of a human vaccine against this pathogen

Regulatory and Safety Requirements for Food Cultures

The increased use of food cultures to ferment perishable raw materials has potentiated the need for regulations to assess and assure the safety of food cultures and their uses. These regulations differ from country to country, all aimed at assuring the safe use of food cultures which has to be guaranteed by the food culture supplier. Here we highlight national differences in regulations and review a list of methods and methodologies to assess the safety of food cultures at strain level, at production, and in the final product

Ability of coagulase negative staphylococci to synthesize nitric oxide: impact of nitric oxide synthase from Staphylococcus xylosus

National audienceCoagulase negative staphylococci (CNS) are frequently isolated from meat products. Among them, the species Staphylococcus xylosus and S. carnosus are used as meat starter cultures. In these products, nitrate and nitrite are added for the development of color. CNS contribute to this development through their nitrate reductase activity that reduces nitrate to nitrite. The chemical reduction of nitrite to nitric oxide (NO) leads to the formation of the stable bright red nitrosomyoglobin, the typical pigment of cured meat products. The safety regarding the use nitrite is questioned because it can react with secondary amines to form unwanted nitrosamines. Some bacteria can synthetize NO from arginine via the nitric oxide synthase (NOS). In all sequenced Staphylococcus genomes, the gene encoding NOS is present. We aimed to determine the role of NOS in S. xylosus. Then, we evaluated the diversity of NOS among CNS strains isolated from meat and assessed their potential to produce NO. A mutant strain deleted from nos (Δnos) was generated in S. xylosus C2a. The ability of this mutant to produce NO was evaluated by the conversion of metmyoglobin to nitrosomyoglobin followed by extraction of the nitrosoheme. In the Δnos mutant, nitrosoheme formation was sharply reduced under limited oxygen conditions, while it was abolished under aerobic conditions. The NOS sequences are highly conserved among the 5 targeted CNS species. A high diversity to form red myoglobin derivatives from metmyoglobin was observed between the species and within the strains. When nitrosoheme is formed, it was always in low amount. The main red myoglobin derivative formed by the strains was oxymyoglobin. This work has allowed to identify a NOS activity within S. xylosus and revealed the capacity of some CNS strains isolated from meat to form nitrosomyoglobin as well as oxmyoglobin

Production de monoxyde d’azote par les staphylocoques à coagulase négative

National audienceLes staphylocoques à coagulase négative (SCN) sont fréquemment isolés des produits carnés. Dans ces produits, il est d’usage de rajouter du nitrate et du nitrite pour le développement de la couleur. Les SCN contribuent à ce développement grâce à leur activité nitrate réductase qui réduit le nitrate en nitrite. Le nitrite est chimiquement réduit en monoxyde d’azote (NO) qui interagit avec le fer de l’hème de la myoglobine pour former de la nitrosomyoglobine, le pigment rouge typique des salaisons. L’utilisation du nitrite dans ces produits est remise en question en raison d’un risque lié à de la formation de composés N-nitrosés tels que les nitrosamines. Certaines bactéries ont la capacité à produire du NO grâce à l’activité de leur NO-synthase (NOS). Le gène codant cette enzyme est présent dans tous les génomes de staphylocoque séquencés. Nos objectifs ont été de déterminer le rôle de la NOS de S. xylosus dans la synthèse de NO, d’évaluer la diversité des NOS parmi des souches de SCN isolées de viande et leur capacité à produire du NO. Un mutant de délétion du gène nos (Δnos) a été construit chez la souche S. xylosus C2a. Sa capacité à produire du NO a été évaluée par conversion de la metmyoglobine en nitrosomyoglobine, suivi de l’extraction et de la mesure du nitrosohème formé, en condition limitée en oxygène et en aérobiose. Un arbre phylogénétique a été réalisé à partir d’un alignement de séquences NOS de 113 souches appartenant à 5 espèces de SCN. La capacité des souches à produire du NO a été évaluée. Chez le mutant S. xylosus Δnos, la formation de nitrosohème était fortement réduite en condition limitée en oxygène et abolie en aérobiose en comparaison de la souche sauvage. La séquence des NOS est très conservée au sein des différentes espèces. Une importante diversité entre les espèces et les souches a été observée quant à leur capacité à former des pigments rouges à partir de la metmyoglobine. Celles qui étaient capables de former de la nitrosomyoglobine et donc du nitrosohème n’en formaient qu’en faible quantité. Le principal pigment rouge formé était de l’oxymyoglobine. Cette étude a permis de caractériser une activité NOS chez une souche de S. xylosus, une espèce couramment utilisée comme ferment carné. La capacité de certaines souches de SCN isolées de viande à former de la nitrosomyoglobine a été révélée ainsi que leur capacité à former de l’oxymyoglobine, un autre pigment rouge

Evidence for nitric oxide synthase activity in staphylococcus xylosus mediating nitrosoheme formation

Staphylococcus xylosus is used as a starter culture in fermented meat products and contributes to color formation by the reduction of nitrate to nitrite. Nitrite is a food additive that is chemically turned to nitric oxide (NO) in meat but its safety has been questioned. The objective of this study was to determine the ability of NO synthase (NOS) of S. xylosus C2a to produce NO. For this purpose, a nos deletion mutant (Δnos) in S. xylosus was constructed and NO production was evaluated in a test based on its ability to form nitrosomyoglobin and nitrosoheme. Production of NO was abrogated in the Δnos mutant under aerobic conditions and reduced about 35-40% comparing to the wild type C2a under limited oxygenation. This mutant was sensitive to oxidative stress. The expression of genes encoding catalase was modulated in the mutant with an up-regulation of katA and a down-regulation of katB and katC. The Δnos mutant displayed high colony pigmentation after prolonged growth on agar medium. Finally, the Δnos mutant showed no growth in minimal medium. Growth was not restored in the minimal medium by complementation with nos, but was restored by either addition of phenylalanine or complementation with pdt, a gene that encodes a prephenate dehydratase involved in phenylalanine biosynthesis and co-transcribed with nos. Our findings clearly demonstrate NOS-mediated NO production in S. xylosus, a meat-associated coagulase-negative Staphylococcus

Patient satisfaction with the colonoscopy procedure: endoscopists overestimate the importance of adverse physical symptoms

BACKGROUND: Colonoscopy is a frequently performed procedure associated with a substantial burden for the patient. Most of what is known about patient satisfaction stems from surveys that target issues doctors believe to be important. It has been noticed that patients often focus on different aspects. AIM: To have patients and endoscopists rate the extent to which each of a list of patient-generated issues-of-concern contributes to patient satisfaction with the colonoscopy procedure. SUBJECTS: A sample of consecutive patients undergoing colonoscopy in a Dutch tertiary teaching hospital and a convenience sample of endoscopists. METHODS: Colonoscopy patients and endoscopists were asked to rate on a five-point Likert scale the importance of 55 items concerning the colonoscopy procedure for patient satisfaction. Items were derived from focus group sessions with colonoscopy patients. Endoscopists were invited to rate the importance of the same set of items from a patient perspective. An analysis was carried out of whether patients and endoscopists rated the importance of items differently. RESULTS: 69 patients and 34 endoscopists completed the questionnaire. The ratings of the endoscopists were significantly different from those of patients (p<0.0001). Endoscopists underestimated the importance of involving patients in decisions, discussing risks and complications, providing the opportunity for substantive questions and offering a comfortable temperature in the examination room. Endoscopists overestimated the importance of adverse physical symptoms, such as pain and abdominal cramps, and the role of the treating doctor. CONCLUSIONS: Endoscopists do not have a good perception of the items that contribute most to patient satisfaction with the colonoscopy procedure. Overcoming this gap may be an essential step towards improving patient satisfaction by targeting those concerns most relevant to patients