Intricate Crosstalk Between Food Allergens, Phages, Bacteria, and Eukaryotic Host Cells of the Gut-skin Axis.

Bacterial and food allergens are associated with immune-mediated food allergies via the gut-skin axis. However, there has been no data on the potential use of phages to rescue this pathological process. A human triple cell co-culture model incorporating colonocytes (T84 cells), macrophages (THP-1 cells), and hepatocytes (Huh7 cells) was established and infected with Pseudomonas aeruginosa PAO1 (P.a PAO1) in the absence or presence of its KPP22 phage in Dulbecco's Modified Eagle's Medium (DMEM), DMEM+ ovalbumin (OVA), or DMEM+ß-casein media. The physiological health of cells was verified by assessing cell viability and Transepithelial electrical resistance (TEER) across the T84 monolayer. The immune response of cells was investigated by determining the secretions of IL-1ß, IL-8, IL-22, and IL-25. The ability of P.a PAO1 to adhere to and invade T84 cells was evaluated. The addition of either OVA or ß-casein potentiated the P.a PAO1-elicited secretion of cytokines. The viability and TEER of the T84 monolayer were lower in the P.a PAO1+OVA group compared to the P.a PAO1 alone and PAO1+ß-casein groups. OVA and ß-casein significantly increased the adherence and invasion of P.a PAO1 to T84 cells. In the presence of the KPP22 phage, these disruptive effects were abolished. These results imply that: (1) food allergens and bacterial toxic effector molecules exacerbate each other's disruptive effects; (2) food allergen and bacterial signaling at the gut-skin mucosal surface axis depend on a network of bacteria-phage-eukaryotic host interactions; and (3) phages are complementary for the evaluation of pathobiological processes that occur at the interface between bacteria, host cellular milieu, and food antigens because phages intervene in P.a PAO1-, OVA-, and ß-casein-derived inflammation.

The life and times of yeasts in traditional food fermentations.

Yeasts are eukaryotic microorganisms which have a long history in the biotechnology of food production, as they have been used since centuries in bread-making or in the production of alcoholic beverages such as wines or beers. Relative to this importance, a lot of research has been devoted to the study of yeasts involved in making these important products. The role of yeasts in other fermentations in association with other microorganisms - mainly lactic acid bacteria - has been relatively less studied, and often it is not clear if yeasts occurring in such fermentations are contaminants with no role in the fermentation, spoilage microorganisms or whether they actually serve a technological or functional purpose. Some knowledge is available for yeasts used as starter cultures in fermented raw sausages or in the production of acid curd cheeses. This review aimed to summarize the current knowledge on the taxonomy, the presence and potential functional or technological roles of yeasts in traditional fermented plant, dairy, fish and meat fermentations.

Genome analysis of the temperate bacteriophage PMBT6 residing in the genome of Bifidobacterium thermophilum MBT94004.

The Siphoviridae phage PMBT6 was identified by transmission electron microscopy in the supernatant of Bifidobacterium thermophilum MBT94004 bioreactor fermentation culture, where it occurred at a moderately high titer. Genome analysis of the bacterial DNA confirmed the presence of this prophage within the genome of the lysogenic host. Under laboratory conditions, the prophage could not be induced by mitomycin C, ultraviolet C irradiation or hydrogen peroxide, suggesting that the prophage was released by spontaneous induction under (yet unknown) bioreactor conditions. Genome sequencing of the virion resulted in a linear, double-stranded DNA molecule of 36,561 bp with a mol% G + C content of 61.7 and 61 predicted open reading frames with low similarity to other Bifidobacterium spp. genomes, confirming that PMBT6 represents a novel temperate phage for this genus.

Safety, functional properties and technological performance in whey-based media of probiotic candidates from human breast milk.

We aimed at isolating and characterising microorganisms present in human breast milk with probiotic potential. In an 8-week postpartum sampling period, two strains of bifidobacteria (Bifidobacterium longum LM7a and Bifidobacterium dentium LM8a') and four strains of lactobacilli were isolated, all during the first 4-week postpartum. B. longum LM7a and B. dentium LM8a', together with four strains previously isolated from breast milk (Bifidobacterium lactis INL1, INL2, INL4 and INL5), were considered for further studies. Susceptibility of the strains to tetracycline, erythromycin, clindamycin, streptomycin, vancomycin and chloramphenicol was evaluated and the isolates exhibited, in general, the same properties as previously reported for bifidobacteria. All isolates showed low hydrophobicity and B. lactis and B. longum strains had satisfactory resistance to gastric digestion and bile shock, but not to pancreatin. B. lactis INL1, B. longum LM7a and B. dentium LM8a' were selected for some comparative technological studies. In particular, B. lactis INL1 displayed technological potential, with satisfactory growth in cheese whey-based media in biofermentor and resistance to freeze-drying, accelerated storage conditions and simulated gastric digestion.

Halomonas nigrificans sp. nov., isolated from cheese.

A Gram-stain-negative, rod-shaped Proteobacteria isolate, MBT G8648T, was obtained from an acid curd cheese called Quargel. The isolate was moderately salt tolerant and motile, with numerous peritrichous flagella. The 16S rRNA gene sequence analysis indicated that the strain belongs to the genus Halomonas, with 98.42 % 16S rRNA gene sequence similarity with Halomonas titanicae BH1T as nearest related neighbour. Further comparative sequence analysis of secA and gyrB genes, as well as physiological and biochemical tests, revealed that this bacterium formed a taxon well-separated from its nearest neighbours and other established Halomonas species. Thus, the strain represents a new species, for which the name Halomonas nigrificans sp. nov. is proposed, with strain MBT G8648T (=LMG 29097T =DSM 105749T) as type strain.

Exploring interplay between bovine milk-derived α-lactalbumin, pathogenic bacteria, and bacteriophages at the molecular interface of inflammation.

There is so far no available data about how the additive, synergistic, or antagonistic effects of the combined form of alpha-lactalbumin (α-La) and bacteriophages might modulate the cellular milieu of the host-pathogen interface. A co-culture of colonocytes and hepatocytes was stimulated with Pseudomonas aeruginosa PAO1 in the presence of KPP22 phage and incubated for 6 hours in medium alone or medium supplemented with bovine milk-origin α-La. The combination of KPP22 phage and α-La significantly inhibited P.a PAO1-elicited secretion of IL-1ß, IL-6, and ICAM-1, which are the mediators and enzymes associated with the inflammatory response to an infectious-inflamed milieu. Cell viability was higher in the P.a PAO1+ KPP22 phage group compared to the P.a PAO1alone group. KPP22 phage and α-La, either alone or in combination, rescued P.a PAO1-induced aberrant PGE1/PGE2 production ratios. The convergence of ingested α-La and phages mitigates pro-inflammatory mediators. α-La leads to an increased sensitivity of opportunistic pathogenic bacteria to phages. Structural, functional, or immunological similarities between ingested α-La and phages play an important role in the mitigation of infection-driven pathobiological processes.

Endogenous Ethanol-producing Bacteria Interference in Pathogen-host Crosstalk.

BACKGROUND AND AIMS: The host micronutrient milieu is a compilation of factors of both endogenous and exogenous origin. This milieu shapes the host's immune responses and can control the inflammatory response of the host when infected. Among vitamins, B12 plays a key role in the defense process because there is intense competition for it between pathogenic invaders and infected host cells. Alcoholic beverages and antibiotics can cause biological (in vivo) interferences that affect pathogenhost crosstalk. Ethanol is known to interfere with the absorption, distribution, and excretion of vitamin B12 in men and animals. However, the molecular mechanisms underlying this backdrop are not fully understood. Here, we explored how Gram-positive ethanol-producing and Gram-negative vitamin B12- producing microbes of the infected milieu interact to influence biomarkers of host cell defense responses in absorbing, digesting, and defensive cells. MATERIAL AND METHODS: We investigated two different cell types of colon and liver origin, hepatic-like Huh7 cells and HT- 29/B6 colon cells. To assess the ability of secreted factors from bacteria to exert influence on co-cultured cell's secretion of host-defense markers in response to invading pathogens, cocultured human colonic HT-29/B6 and human hepatic Huh-7 (hereafter Huh7) cells were stimulated or not with Klebsiella pneumoniae 52145 for 24 h in the presence or absence of either Weissella confusa strain NRRL-B-14171 (as a Gram-positive producer of ethanol), Limosilactobacillus reuteri 20016 (as a Gram-positive producer of vitamin B12), or Pseudomonas nitroreducens 1650 (as a Gram-negative producer of vitamin B12). After stimulation, molecular functional biomarkers of host cell defense responses including total MMP-1, lysozyme activity, ALP, and IL-25 were measured. RESULTS: While simultaneously reducing IL-25 secretion, Kp52145 alone significantly elicited MMP-1, lysozyme, and ALP secretion from co-cultured cells, as compared to no treatment. When compared with Kp 52145 stimulation alone, Pn1650 significantly potentiated MMP-1 and lysozyme secretions from Kp 52145-stimulated co-cultured cells by 29.7% and 67.4%, respectively. Simultaneously, a potentiated suppression (an overall decrease of 77.3%) in IL-25 secretion occurred 24 hours after Kn52145 plus Pn1650 administration. Compared to Kp52145-stimulation alone, treatment with W. confusa NRRL-B-14171 and Kp52145-stimulated co-cultured cells was associated with significant additive induction of MMP-1 and lysozyme secretions. However, compared to Kp52145-stimulation alone, W. confusa NRRL-B-14171 treatment significantly potentiated Kp52145-induced suppression of IL-25. Using the same condition as mentioned above and compared to Kp52145-stimulation alone, L. reuteri 20016 treatment altered the secretion pattern in response to Kp52145: L. reuteri 20016-treated cells displayed less aversive responses towards Kp52145, suggesting that L. reuteri 20016 modulation may act differently on Kp52145 - induced signaling. CONCLUSION: Gram-negative and Gram-positive vitamin B12- producing bacteria differently affect the secretion of key immune biomarkers in co-cultured HT-29/B6 and Huh7 cells following exposure to Kp52145. Ethanol-producing bacteria additively potentiate pathogenicity and inflammatory responses upon infection. To confirm the biological consequences of these effects on human gut microbiota and health, further studies are warranted, incorporating ex vivo studies of human colon samples and host biomarkers such as cytohistological, molecular, or biochemical measurements.

Oral Mucosal In Vitro Cell Culture Model to Study the Effect of Fructilactobacillus Phage on the Interplay between Food Components and Oral Microbiota.

BACKGROUND AND AIMS: Dietary habits, food, and nutrition-associated oral dysbiosis lead to the formation of microbial biofilm, which affects the overall health of an individual by promoting systemic diseases like cardiovascular disease, immunological disorders, and diabetes. Today's diets contain a variety of fermentable carbohydrates, including highly processed starch and novel synthetic carbohydrates such as oligofructose, sucralose, and glucose polymers. These constitute risk factors in the initiation and progression of oral dysbiosis. Oral, lung and gut microbiomes are interlinked with each other via direct and indirect ways. It is unknown whether or not lactobacilli and Lactobacillus phages are able to rescue dysbiotic effects by decreasing the uptake into the cells of excess simple sugars and their derivatives present within the digestive tract. MATERIALS AND METHODS: Using transwell cell culture plate inserts, six groups of in vitro co-cultured TR146 and HepG2 cells, grown in DMEM medium either with or without sucrose (10 % v/v), were treated with 1) PBS, 2) Fructilactobacillus sanfranciscensis (F.s) H2A, 3) F.s H2A and sucrose, 4) F.s H2A plus sucrose plus phage EV3 lysate, 5) F.s H2A plus sucrose plus phage EV3 supernatant, and 6) F.s H2A plus sucrose plus phage EV3 particles. The pH of the culture medium (indicating lactic acid production) and key oral biomarkers, including cytokines (IL-1ß and IL-6), inflammatory chemokines (e.g., CXCL8 and CCL2), and homeostatic chemokines (e.g., CXCL4 and CCL18) were measured. RESULTS: Excess sucrose significantly enhanced inflammatory signal molecules (e.g., IL-1ß, IL-6, and CCL2) secretion, concomitant with the enhancement of intracellular triglycerides in co-cultured HepG2 cells. Co-culture with F.s H2A decreased the sucrose-induced release of inflammatory signal molecules from co-cultured cells, these effects being abolished by F.s phage EV3. CONCLUSION: This study shows that Lactobacillus phages apparently influence the interplay between food components, oral microbiota, and the oral cellular milieu, at least in part by affecting the microbial uptake of excess free simple sugars from the oral milieu. To confirm the biological consequences of these effects on human oral microbiota and health, further studies are warranted, incorporating ex vivo studies of human dental plaque biofilms and host biomarkers, such as cytohistological, molecular, or biochemical measurements.

Exploring the Interplay between Nutrients, Bacteriophages, and Bacterial Lipases in Host- and Bacteria-mediated Pathogenesis.

BACKGROUND AND AIMS: Pathogenic bacteria and host cells counteract or neutralize each other's effect in two fundamental ways: Direct invasion and secretion of various substances. Among these, lipases secreted by pathogenic bacteria and host cell lysozyme are key actors. Secreted lipases from pathogenic bacterial are suggested as a key player in the pathogen-host interaction. Among the gut microbial energy sources, glucose and fats have been referred to as one of the best inducers and substrates for bacterial lipases. Enrichment of bacterial growth medium with extra glucose or oil has been shown to induce lipase production in pathogenic bacteria. More recently, research has focused on the role of human gut phage alterations in the onset of dysbiosis because the bacteria-phage interactions can be dramatically affected by the nutrient milieu of the gut. However, the reciprocal role of bacterial lipases and phages in this context has not been well studied and there is no data available about how high glucose or fat availability might modulate the cellular milieu of the pathogenic bacteria-phageeukaryotic host cell interface. The purpose of this study was to evaluate the immunologic outcome of pathogenic bacteria-phage interaction under normal, high glucose, and high butter oil conditions to understand how nutrient availability affects lipase activity in pathogenic bacteria and, ultimately, the eukaryotic host cell responses to pathogenic bacteria-phage interaction. MATERIALS AND METHODS: 10 groups of co-cultured T84 and HepG2 cells were treated with Pseudomonas aeruginosa strain PAO1 (P.a PAO1) in the presence and absence of its KPP22 phage and incubated in three different growth media (DMEM, DMEM + glucose and DMEM + butter oil). Structural and physiological (barrier function and cell viability), inflammatory (IL-6 and IL-8), metabolic (glucose and triglycerides), and enzymatic (lipases and lysozyme) parameters were determined. RESULTS: Excess glucose or butter oil enhanced additively extracellular lipase activity of P.a PAO1. Excess glucose or butter oil treatments also magnified P. a PAO1- induced secretion of inflammatory signal molecules (IL-1ß, IL-6) from co-cultured cells, concomitant with the enhancement of intracellular triglycerides in co-cultured HepG2 cells, these effects being abolished by phage KPP22. CONCLUSION: The results of the present study imply that KPP22 phage influences the interplay between food substances, gut bacterial lipases, and the gut cellular milieu. This can be applied in two-way interaction: by affecting the microbial uptake of excess free simple sugars and fats from the gut milieu leading to decreased bacterial lipases and by modulating the immune system of the intestinal -liver axis cells. Further studies are needed to see if the biological consequences of these effects also occur in vivo.

Effects of ad libitum Free-Choice Access to Freshly Squeezed Domestic White Asparagus Juice on Intestinal Microbiota Composition and Universal Bio-Markers of Immuno-Metabolic Homeostasis and General Health in Middle-Aged Female and Male C57BL/6 Mice.

BACKGROUND: Asparagus contains different bioactive and volatile components including pyrazines, sulphur-containing compounds, and polyphenols. Asparagus juice is a new low-calorie LAB-containing natural juice product, the usage of which is expanding. Pyrazines and sulphur-containing compounds are degraded by bacteria on one hand, but on the other hand, dietary polyphenols prevent human colorectal diseases as modulators of the composition and/or activity of gut microbiota. However, the utility of these asparagus compounds for reversal of age-associated microbial dysbiosis and the immunometabolic disorders that dysbiosis incites body inflammatory reactions was not much explored so far. Hence, using middle-aged mice, we conducted the current study to verify the effect of freshly squeezed domestic white asparagus juice on the biomarkers reflecting immuno-metabolic pathways linking age-related dysbiosis and metabolic events. MATERIALS AND METHODS: Thirty-two conventional Harlan Laboratories C57BL/6 mice aged between 11-12 months were randomly divided into two groups (n=16). Mice in control group 1 received sterile tap water. Animals in group 2 had 60 days ad libitum free-choice access to sterile tap water supplemented with 5% (v/v) freshly squeezed domestic white asparagus juice. Clinical signs of general health, hydration, and inflammation were monitored daily. Caecal content samples were analysed by qPCR for microbial composition. Histology of relevant organs was carried out on day 60 after sacrificing the mice. Universal markers of metabolic- and liver function were determined in serum samples. Caecal SCFAs contents were measured using HPLC. RESULTS: Overall, no significant differences in general health or clinical signs of inflammation between the two groups were observed. The liver to body weight ratio in asparagus juice-drank mice was lowered. The qPCR quantification showed that asparagus juice significantly decreased the caecal Clostridium coccoides group while causing an enhancement in Clostridium leptum, Firmicutes, and bifidobacterial groups as well as total caecal bacterial count. Asparagus juice significantly elevated the caecal contents of SCFAs. Enhanced SCFAs (acetate, butyrate, and propionate) in mice receiving asparagus juice, however, did coincide with altered lipid levels in plasma or changes in the abundance of relevant bacteria for acetate-, butyrate-, and propionate production. DISCUSSION: To the best of our knowledge, this is the first study aiming at evaluating the effect of freshly squeezed German domestic white asparagus juice on universal markers of metabolic- and liver function in middle- aged mice and the role of gut microbiota in this regard. The effectiveness of asparagus juice to improve metabolism in middle-aged mice was associated with alterations in intestinal microbiota but maybe also due to uptake of higher amounts of SCFAs. CONCLUSION: Hence, the key signal pathways corresponding to improved immune-metabolic homeostasis will be an important research scheme in the future.

Isolation and Characterization of Potential Starter Cultures from the Nigerian Fermented Milk Product nono.

Nono, an important traditional fermented dairy food produced from cow's milk in Nigeria, was studied for microbial diversity and for starter culture development for industrial production. On the basis of a polyphasic approach, including phenotypic and genotypic methods such as 16S rRNA gene sequencing, repetitive element PCR (rep-PCR) fingerprinting metagenomics, and whole genome sequencing, we identified Lactobacillus (Lb.) helveticus, Limosilactobacillus (L.) fermentum, Lb. delbrueckii, and Streptococcus (S.) thermophilus as predominant bacterial species involved with milk fermentation during traditional nono production in Nigeria, while the predominant yeast species in nono was identified as Saccharomyces cerevisiae. Using metagenomics, Shigella and potential pathogens such as enterobacteria were detected at low levels of abundance. Strains of the predominant lactic acid bacteria (LAB) were selected for starter cultures combination on the basis of their capacities for rapid growth in milk and reduction of pH below 4.5 and their gelling characteristic, which was demonstrated noticeably only by the S. thermophilus strains. Whole genome sequence analysis of selected bacterial strains showed the largest assembled genome size to be 2,169,635 bp in Lb. helveticus 314, while the smallest genome size was 1,785,639 bp in Lb. delbrueckii 328M. Genes encoding bacteriocins were not detected in all the strains, but all the LAB possessed genes potentially involved in diacetyl production and citrate metabolism. These bacteria isolated from nono can thus be used to improve the microbial safety quality of nono in Nigeria, in addition to improving technological parameters such as gelling viscosity, palatability, and product consistency.

Study on the additive protective effect of PGLYRP3 and Bifidobacterium adolescentis Reuter 1963 on severity of DSS-induced colitis in Pglyrp3 knockout (Pglyrp3 -/-) and wild-type (WT) mice.

BACKGROUND AND AIMS: Pglyrp3 is a bactericidal innate immunity protein known to sustain the habitual gut microbiome and protect against experimental colitis. Intestinal inflammation and metaflammation are commonly associated with a marked reduction of commensal bifidobacteria. Whether Pglyrp3 and bifidobacteria interact synergistically or additively to alleviate metaflammation is unknown. We investigated the extent to which Pglyrp3 and bifidobacteria regulate metaflammation and gut bacterial dysbiosis in DSS-induced mouse models of intestinal inflammation. MATERIAL & METHODS: 8-10 weeks old male mice were used. In both WT and Pglyrp3 -/- experiments, the mice were randomly divided into three groups of 16 mice per group: (1) a control group receiving sterile tap water, (2) an experimental group receiving sterile tap water supplemented with only 5% DSS, and (3) an experimental group receiving sterile tap water supplemented with 5% DSS and 1 × 109 CFU/ml of Bifidobacterium adolescentis (B.a.) for 7 days. Wild-type (WT) littermates of the respective gene (i.e. Pglyrp3) were used as controls throughout the study. Clinical signs of general health and inflammation were monitored daily. Faecal pellet samples were analysed by qRT-PCR for microbial composition. Histology of relevant organs was carried out on day 8. Metabolic parameters and liver inflammation were determined in serum samples. RESULTS: Intestinal inflammation in mice of group 2 were significantly increased compared to those of control group 1. There was a significant difference in mean scores for inflammation severity between DSS-treated WT and DSS-treated Pglyrp3 -/- mice. Buildup of key serum metabolic markers (cholesterol, triglyceride and glucose) was set off by colonic inflammation. qRT-PCR quantification showed that DSS significantly decreased the Clostridium coccoides and Bifidobacterium cell counts while increasing those of Bacteroides group in both WT and Pglyrp3 -/- mice. These manifestations of DSS-induced dysbiosis were significantly attenuated by feeding B.a. Both the local and systemic ill-being of the mice alleviated when they received B.a. DISCUSSION: This study shows that Pglyrp3 facilitates recognition of bifidobacterial cell wall-derived peptidoglycan, thus leading additively to a reduction of metaflammation through an increase in the number of bifidobacteria, which were able to mitigate intestinal immunopathology in the context of Pglyrp3 blockade.

Effect of Oral Administration of Weissella confusa on Fecal and Plasma Ethanol Concentrations, Lipids and Glucose Metabolism in Wistar Rats Fed High Fructose and Fat Diet.

BACKGROUND AND PURPOSE: In previous investigations, Weissella confusa was shown to lack the metabolic pathway from fructose to mannitol and to produce ethanol when cultivated in the presence of fructose. Hence, we assessed the effect of oral administration of W. confusa (strain NRRL-B-14171) on blood and fecal ethanol concentrations, glucose and lipid metabolism and traits of the metabolic syndrome in Wistar rats (n=27) fed diets with two different fat and fructose levels and with or without the addition of W. confusa during a total intervention time of 15 weeks (105 days). MATERIALS AND METHODS: From week 1 to 6, rats were given a medium fructose and fat (MFru-MF) diet containing 28% fructose and 10% fat without the addition of W. confusa (control group, n=13) or mixed with 30 g per kg diet of lyophilized W. confusa (10.56 ± 0.20 log CFU/g; W. confusa group, n=14). From week 7 to 15, the percentage of dietary fructose and fat in the control and W. confusa group was increased to 56% and 16%, respectively (high fructose-high fat (HFru-HF) diet). RESULTS: In HFru-HF-fed rats, W. confusa was detected in feces, regardless of whether W. confusa was added to the diet or not, but not in rats receiving the MFru-MF diet without added W. confusa or in an additional control group (n=10) fed standard rat food without fructose, increased fat content and W. confusa. This indicates that fecal W. confusa may be derived from orally administered W. confusa as well as - in the case of high fructose and fat intake and obesity of rats - from the intestinal microbiota. As shown by multifactorial ANOVA, blood ethanol, the relative liver weight, serum triglycerides, and serum cholesterol as well as fecal ethanol, ADH, acetate, propionate and butyrate, but not lactate, were significantly higher in the W. confusa - compared to the control group. DISCUSSION: This is the first in vivo trial demonstrating that heterofermentative lactic acid bacteria lacking the mannitol pathway (like W. confusa) can increase fecal and blood ethanol concentrations in mammals on a high fructose-high fat diet. This may explain why W. confusa resulted in hyperlipidemia and may promote development of NAFLD in the host.

Draft Genome Sequence of Citrobacter gillenii MBT-C3, Isolated from Lamb's Lettuce.

The genome of the streptomycin-resistant Citrobacter gillenii strain MBT-C3, isolated from lamb's lettuce in Germany, was sequenced. Sequence analysis showed the assembled draft genome size to be 5,167,205 bp, containing a predicted total of 5,011 protein-encoding genes, 8 rRNAs, and 71 tRNAs.

Diversity and Antibiotic Susceptibility of Acinetobacter Strains From Milk Powder Produced in Germany.

Forty-seven Acinetobacter spp. isolates from milk powder obtained from a powdered milk producer in Germany were investigated for their antibiotic resistance susceptibilities, in order to assess whether strains from food harbor multiple antibiotic resistances and whether the food route is important for dissemination of resistance genes. The strains were identified by 16S rRNA and rpoB gene sequencing, as well as by whole genome sequencing of selected isolates and their in silico DNA-DNA hybridization (DDH). Furthermore, they were genotyped by rep-PCR together with reference strains of pan-European groups I, II, and III strains of Acinetobacter baumannii. Of the 47 strains, 42 were identified as A. baumannii, 4 as Acinetobacter Pittii, and 1 as Acinetobacter calcoaceticus based on 16S rRNA gene sequencing. In silico DDH with the genome sequence data of selected strains and rpoB gene sequencing data suggested that the five non-A. baumannii strains all belonged to A. pittii, suggesting that the rpoB gene is more reliable than the 16S rRNA gene for species level identification in this genus. Rep-PCR genotyping of the A. baumannii strains showed that these could be grouped into four groups, and that some strains clustered together with reference strains of pan-European clinical group II and III strains. All strains in this study were intrinsically resistant toward chloramphenicol and oxacillin, but susceptible toward tetracycline, tobramycin, erythromycin, and ciprofloxacin. For cefotaxime, 43 strains (91.5%) were intermediate and 3 strains (6.4%) resistant, while 3 (6.4%) and 21 (44.7%) strains exhibited resistance to cefepime and streptomycin, respectively. Forty-six (97.9%) strains were susceptible to amikacin and ampicillin-sulbactam. Therefore, the strains in this study were generally not resistant to the clinically relevant antibiotics, especially tobramycin, ciprofloxacin, cefepime, and meropenem, suggesting that the food route probably poses only a low risk for multidrug resistant Acinetobacter strains or resistance genes.

Isolation and Characterization of Lactic Acid Bacteria from Fermented Goat Milk in Tajikistan.

The lactobacilli associated with a fermented goat milk product from Tajikistan were isolated to characterize their technological properties and antibiotic resistances in order to assess their suitability for development as starter cultures. In this study, twenty three strains were identified by 16S rRNA sequencing as typical dairy-associated lactic acid bacterial strains, i.e. L. plantarum, L. pentosus, L. delbrueckii, L. helveticus and L. paracasei. These strains were generally susceptible to most antibiotics tested in this study and this allowed a selection of strains as safe starters. The draft genomes of four representative strains were sequenced and the number of contigs of the four assembled genomes ranged from 51 to 245 and the genome sizes ranged from 1.75 to 3.24 Mbp. These representative strains showed differences in their growth behavior and pH-reducing abilities in in vitro studies. The co-inoculation of these Lactobacillus spp. strains together with a yeast Kluyveromyces marxianus MBT-5698, or together with the yeast and an additional Streptococcus thermophilus MBT-2, led to a pH reduction to 3.4 after 48 h. Only in the case of fermentation inoculated with the co-culture, the viscosity of the milk increased noticeably. In contrast, fermentations with single strains did not lead to gelation of the milk or to a decrease in the pH after 24h. The results of this study provide a comprehensive understanding of the predominant lactobacilli related to Tajikistani fermented milk products.

Molecular identification and differentiation of Brevibacterium species and strains.

Amplified ribosomal DNA restriction enzyme analysis (ARDRA), pulsed field gel electrophoresis (PFGE) and ribotyping were used to differentiate among 24 strains of Brevibacterium linens, Brevibacterium casei and Brevibacterium epidermidis obtained from type culture collections or isolated from various smear ripened cheeses. ARDRA was applied to the 16S rDNA. B. linens was shown to be a quite heterogenic group with 2 to at least 4 copies of rrn operons per strain with aberrant nucleotide sequences. AccI gave genus specific restriction patterns and was used to separate Brevibacterium from Corynebacterium species. The expected species specificity of TaqI applied to B. linens type culture strains, but not to all strains isolated from cheese. By AvaI restriction, B. casei and B. linens were differentiated from B. epidermidis and the orange pigmented Arthrobacter casei, a new species of coryneform bacteria; by XmnI restriction, B. linens and B. epidermidis were differentiated from B. casei. One of 4 B. linens genotypes could not be distinguished from B. casei by this method. Here, the typical orange B. linens pigments were used for classification, which was confirmed by partial sequencing of the 16S rDNA.

Influence of fermentation on glucosinolates and glucobrassicin degradation products in sauerkraut.

A systematic investigation was carried out on the influence of fermentation on glucosinolates and their degradation products from fresh raw cabbage, throughout fermentation at 20 °C and storage at 4 °C. Glucosinolates were degraded dramatically between Day 2 and 5 of fermentation and by Day 7 there was no detectable amount of glucosinolates left. Fermentation led to formation of potential bioactive compounds ascorbigen (13.0 µmol/100 g FW) and indole-3-carbinol (4.52 µmol/100g FW) with their higher concentrations from Day 5 to Day 9. However, during storage indole-3-carbinol slowly degraded to 0.68 µmol/100 g FW, while ascorbigen was relatively stable from Week 4 until Week 8 at 6.78 µmol/100 g FW. In contrast, the content of indole-3-acetonitrile decreased rapidly during fermentation from 3.6 to 0.14 µmol/100 g FW. The results imply a maximum of health beneficial compounds after fermentation (7-9 days) in contrast to raw cabbage or stored sauerkraut.

Ethanol Production by Selected Intestinal Microorganisms and Lactic Acid Bacteria Growing under Different Nutritional Conditions.

To gain some specific insight into the roles microorganisms might play in non-alcoholic fatty liver disease (NAFLD), some intestinal and lactic acid bacteria and one yeast (Anaerostipes caccae, Bacteroides thetaiotaomicron, Bifidobacterium longum, Enterococcus fecalis, Escherichia coli, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus plantarum, Weissella confusa, Saccharomyces cerevisiae) were characterized by high performance liquid chromatography for production of ethanol when grown on different carbohydrates: hexoses (glucose and fructose), pentoses (arabinose and ribose), disaccharides (lactose and lactulose), and inulin. Highest amounts of ethanol were produced by S. cerevisiae, L. fermentum, and W. confusa on glucose and by S. cerevisiae and W. confusa on fructose. Due to mannitol-dehydrogenase expressed in L. fermentum, ethanol production on fructose was significantly (P < 0.05) reduced. Pyruvate and citrate, two potential electron acceptors for regeneration of NAD(+)/NADP(+), drastically reduced ethanol production with acetate produced instead in L. fermentum grown on glucose and W. confusa grown on glucose and fructose, respectively. In fecal slurries prepared from feces of four overweight volunteers, ethanol was found to be produced upon addition of fructose. Addition of A. caccae, L. acidophilus, L. fermentum, as well as citrate and pyruvate, respectively, abolished ethanol production. However, addition of W. confusa resulted in significantly (P < 0.05) increased production of ethanol. These results indicate that microorganisms like W. confusa, a hetero-fermentative, mannitol-dehydrogenase negative lactic acid bacterium, may promote NAFLD through ethanol produced from sugar fermentation, while other intestinal bacteria and homo- and hetero-fermentative but mannitol-dehydrogenase positive lactic acid bacteria may not promote NAFLD. Also, our studies indicate that dietary factors interfering with gastrointestinal microbiota and microbial metabolism may be important in preventing or promoting NAFLD.

Produce from Africa's Gardens: Potential for Leafy Vegetable and Fruit Fermentations.

A rich variety of indigenous fruits and vegetables grow in Africa, which contribute to the nutrition and health of Africa's populations. Fruits and vegetables have high moisture and are thus inherently prone to accelerated spoilage. Food fermentation still plays a major role in combating food spoilage and foodborne diseases that are prevalent in many of Africa's resource disadvantaged regions. Lactic acid fermentation is probably the oldest and best-accepted food processing method among the African people, and is largely a home-based process. Fermentation of leafy vegetables and fruits is, however, underutilized in Africa, although such fermented products could contribute toward improving nutrition and food security in this continent, where many are still malnourished and suffer from hidden hunger. Fermentation of leafy vegetables and fruits may not only improve safety and prolong shelf life, but may also enhance the availability of some trace minerals, vitamins and anti-oxidants. Cassava, cow-peas, amaranth, African nightshade, and spider plant leaves have a potential for fermentation, as do various fruits for the production of vinegars or fruit beers and wines. What is needed to accelerate efforts for production of fermented leaves and vegetables is the development of fermentation protocols, training of personnel and scale-up of production methods. Furthermore, suitable starter cultures need to be developed and produced to guarantee the success of the fermentations.