Bacterial growth dynamics and corresponding metabolite levels in the extraction area of an Austrian sugar beet factory using antimicrobial treatment.

BACKGROUND: During the manufacture of sucrose from sugar beet, different microorganisms originating from the plant material as well as from the soil enter the process. Due to the formation of polysaccharide-based slimes, these contaminants may induce several adverse effects such as filtration problems during juice purification. Certain microorganisms also metabolize sucrose, leading to product losses with financial consequences. To better understand and to prevent these negative effects, the aim of the study was to investigate the evolution of relevant bacterial groups, including their metabolites appearing during the extraction process. For this purpose, one production cycle was monitored to identify the major contamination steps and to clarify how they relate to the processing conditions. Traditionally, different antimicrobial agents such as formaldehyde, sulfur dioxide, hypochlorous acid, sodium hypochlorite, and chlorine dioxide have been added to inhibit microbial growth. In the present study, a rosin-based product derived from pine trees was applied as an alternative to those substances. RESULTS: Press water, raw juice, and mid-tower juice were identified as being highly contaminated with bacteria, and processing conditions such as time, temperature and pH level significantly influenced bacterial levels and the corresponding metabolites. Among the contaminants identified, lactic acid bacteria, and mesophilic and thermophilic aerobic bacteria played a dominant role, whereas lactic acid, acetic acid, butyric acid, and ethanol were identified as typical metabolites. CONCLUSION: Bacterial growth during production could be reduced by shock dosing of the rosin-based material in the extraction area. © 2020 Society of Chemical Industry.

Fructophilic Lactic Acid Bacteria, a Unique Group of Fructose-Fermenting Microbes.

Fructophilic lactic acid bacteria (FLAB) are a recently discovered group, consisting of a few Fructobacillus and Lactobacillus species. Because of their unique characteristics, including poor growth on glucose and preference of oxygen, they are regarded as "unconventional" lactic acid bacteria (LAB). Their unusual growth characteristics are due to an incomplete gene encoding a bifunctional alcohol/acetaldehyde dehydrogenase (adhE). This results in the imbalance of NAD/NADH and the requirement of additional electron acceptors to metabolize glucose. Oxygen, fructose, and pyruvate are used as electron acceptors. FLAB have significantly fewer genes for carbohydrate metabolism than other LAB, especially due to the lack of complete phosphotransferase system (PTS) transporters. They have been isolated from fructose-rich environments, including flowers, fruits, fermented fruits, and the guts of insects that feed on plants rich in fructose, and are separated into two groups on the basis of their habitats. One group is associated with flowers, grapes, wines, and insects, and the second group is associated with ripe fruits and fruit fermentations. Species associated with insects may play a role in the health of their host and are regarded as suitable vectors for paratransgenesis in honey bees. Besides their impact on insect health, FLAB may be promising candidates for the promotion of human health. Further studies are required to explore their beneficial properties in animals and humans and their applications in the food industry.

Immunogold Nanoparticles for Rapid Plasmonic Detection of C. sakazakii.

Cronobacter sakazakii is a foodborne pathogen that can cause a rare, septicemia, life-threatening meningitis, and necrotizing enterocolitis in infants. In general, standard methods for pathogen detection rely on culture, plating, colony counting and polymerase chain reaction DNA-sequencing for identification, which are time, equipment and skill demanding. Recently, nanoparticle- and surface-based immunoassays have increasingly been explored for pathogen detection. We investigate the functionalization of gold nanoparticles optimized for irreversible and specific binding to C. sakazakii and their use for spectroscopic detection of the pathogen. We demonstrate how 40-nm gold nanoparticles grafted with a poly(ethylene glycol) brush and functionalized with polyclonal antibodies raised against C. sakazakii can be used to specifically target C. sakazakii. The strong extinction peak of the Au nanoparticle plasmon polariton resonance in the optical range is used as a label for detection of the pathogens. Individual binding of the nanoparticles to the C. sakazakii surface is also verified by transmission electron microscopy. We show that a high degree of surface functionalization with anti-C. sakazakii optimizes the detection and leads to a detection limit as low as 10 CFU/mL within 2 h using a simple cuvette-based UV-Vis spectrometric readout that has great potential for further optimization.

Fermentability of a Novel Galacto-Oligosaccharide Mixture by Lactobacillus spp. and Bifidobacterium spp.

This study aimed to investigate the specific growth stimulation of certain desired intestinal bacteria by a novel galacto-oligosaccharide mixture, which was produced with a ß-galactosidase from a potential probiotic Lactobacillus isolate that contained mainly oligosaccharides of ß-1,3 and ß-1,6 glycosidic linkages (termed Lb-GOS) using single-strain fermentations. The composition of this Lb-GOS mixture was 33.5% disaccharides, 60.5% trisaccharides, 4.8% tetrasaccharides, and 1.0% pentasaccharides with a negligible amount of monosaccharides, lactose, and lactobionic acid (0.3%). Eight Lactobacillus spp. strains and three Bifidobacterium spp. strains were used in single-strain fermentations to determine the fermentation activity scores of this Lb-GOS preparation compared to two commercially available prebiotic mixtures, 4'GOS-P and Vivinal GOS (V-GOS). The highest scores were obtained when L. reuteri Lb46 and the two Bifidobacterium strains, B. animalis subsp. lactis Bif1 and Bif3, were grown on these galacto-oligosaccharide mixtures. In addition, the Lb-GOS mixture was found to have higher fermentation activity scores; hence, it stimulated the growth of these probiotic strains more than 4'GOS-P and V-GOS, which may be attributed to the different glycosidic linkage types that are found in the Lb-GOS mixture compared to the other two commercial preparations. These findings suggested that the Lb-GOS mixture that is described in this work should be of interest for the formulations of new carbohydrate-based functional food ingredients.

Disintegration of the agricultural by-product wheat bran under subcritical conditions.

BACKGROUND: The disintegration of destarched wheat bran in water and sulfuric acid (pH 3) under subcritical conditions (275-300 °C) and at short reaction times (1-4 min) was investigated. A cascade process comprising a stepwise separation of the liquid was applied to reduce the formation of undesired degradation products. RESULTS: The highest degree of biomass disintegration (67% dry mass solubilization) was achieved by application of a cascade process at 275 °C (pH 3). Regarding the dissolution of carbohydrates (monomeric and oligomeric form), the total glucose yields remained below 60%, while the total xylose and arabinose yields were about 76% and 67%. Approximately 74% of the protein and 95% of the mineral fraction could be extracted. The application of the cascade process enabled a substantially reduced formation of degradation products. CONCLUSION: When operating hydrothermally and subcritically in order to avoid some problematic aspects of a biorefinery, an extensive disintegration and monomerization of wheat bran and its constituents remains difficult even under the tested conditions (300 °C, pH 3). However, the applied cascade process proved to be useful to increase the yields and to substantially reduce the formation of undesired degradation products. Despite this fact, increased water consumption has to be conceded. © 2018 Society of Chemical Industry.

Celeribacter persicus sp. nov., a polycyclic-aromatic-hydrocarbon-degrading bacterium isolated from mangrove soil.

A Gram-stain-negative, mesophilic bacterial strain, designated SBU1T, which degrades polycyclic aromatic hydrocarbons was isolated from the sediments of the mangrove forests of Nayband Bay in the Iranian Persian Gulf during a bioremediation experiment. The 16S rRNA gene sequence of strain SBU1T exhibited highest similarities with Celeribacter indicus P73T (98.52%) and Celeribacter neptunius H 14T (97.05%). Phylogenetic analysis, based on 16S rRNA gene sequences, demonstrated that strain SBU1T fell within a cluster consisting of the type strains of species of the genus Celeribacter and formed a stable clade with C. indicus P73T in trees generated with three algorithms. The fatty acid profile of strain SBU1T consisted of the major fatty acids C18:1ω7c/ω6c and C18:1ω7c 11-methyl. The major compounds in the polar lipid profile were one phosphatidylglycerol and four unidentified phospholipids. The quinone system exclusively comprised ubiquinone (Q-10). The DNA G+C content was 60.4 mol%. A combination of phylogenetic analysis, DNA-DNA hybridization estimation, average nucleotide identity results and differential phenotypic and chemotaxonomic characteristics demonstrated that strain SBU1T could be distinguished from its close relatives. Therefore, strain SBU1T is considered to represent a novel species of the genus Celeribacter for which the name Celeribacter persicus sp. nov. is proposed. The type strain is SBU1T (=MCCC 1A00672T=DSM 100434T).

The intestinal microbiota of piglets fed with wheat bran variants as characterised by 16S rRNA next-generation amplicon sequencing.

The intestinal microbiota of piglets fed with a Control diet low in dietary fibre and modified wheat bran variants as an additional source of insoluble dietary fibre was characterised. In this context, variances in the microbiota of three different gut segments were assessed. Wheat bran was either included in its native form or modified by fermentation and extrusion before added at 150 g/kg to a basal diet for 48 piglets (12 animals per treatment). Total DNA was extracted from digesta samples from the jejunum, the end of the ileum and the colon ascendens. Samples were prepared accordingly for subsequent sequencing with the Illumina MiSeq. The obtained results revealed distinct location-specific differences in microbial composition. While Firmicutes were most predominant in all three gut segments, Bacteroidetes were additionally found in the colon at high abundance. The parameters of alpha and beta diversity analysis showed significant differences (p < 0.01) between the colon and the other two gut segments. Specialised bacterial groups like Prevotella and Ruminococcaceae were among the most predominant ones found in the colon, as they possess cellulolytic properties to degrade (at least partially) non-starch polysaccharides, while their abundance was negligible in the jejunum and the ileum. Conversely, the genera Lactobacillus, Bifidobacterium and Veillonella, for example, were among the most predominant groups in the jejunum and ileum, while in the colon they were hardly found. Although statistical taxonomical evaluation, following p-value correction, did not reveal pronounced differences in abundance related to bran modification, alpha and beta diversity analysis showed an influence regarding the various feeding strategies applied. Based on these findings, a more in-depth view on intestinal microbial composition within the gastrointestinal tract of young pigs fed with low- and high-fibre diets was generated.

Rhodococcus erythropolis MTHt3 biotransforms ergopeptines to lysergic acid.

BACKGROUND: Ergopeptines are a predominant class of ergot alkaloids produced by tall fescue grass endophyte Neotyphodium coenophialum or cereal pathogen Claviceps purpurea. The vasoconstrictive activity of ergopeptines makes them toxic for mammals, and they can be a problem in animal husbandry. RESULTS: We isolated an ergopeptine degrading bacterial strain, MTHt3, and classified it, based on its 16S rDNA sequence, as a strain of Rhodococcus erythropolis (Nocardiaceae, Actinobacteria). For strain isolation, mixed microbial cultures were obtained from artificially ergot alkaloid-enriched soil, and provided with the ergopeptine ergotamine in mineral medium for enrichment. Individual colonies derived from such mixed cultures were screened for ergotamine degradation by high performance liquid chromatography and fluorescence detection. R. erythropolis MTHt3 converted ergotamine to ergine (lysergic acid amide) and further to lysergic acid, which accumulated as an end product. No other tested R. erythropolis strain degraded ergotamine. R. erythropolis MTHt3 degraded all ergopeptines found in an ergot extract, namely ergotamine, ergovaline, ergocristine, ergocryptine, ergocornine, and ergosine, but the simpler lysergic acid derivatives agroclavine, chanoclavine, and ergometrine were not degraded. Temperature and pH dependence of ergotamine and ergine bioconversion activity was different for the two reactions. CONCLUSIONS: Degradation of ergopeptines to ergine is a previously unknown microbial reaction. The reaction end product, lysergic acid, has no or much lower vasoconstrictive activity than ergopeptines. If the genes encoding enzymes for ergopeptine catabolism can be cloned and expressed in recombinant hosts, application of ergopeptine and ergine degrading enzymes for reduction of toxicity of ergot alkaloid-contaminated animal feed may be feasible.

Comparison of homo- and heterofermentative lactic acid bacteria for implementation of fermented wheat bran in bread.

Despite its potential health benefits, the integration of wheat bran into the food sector is difficult due to several adverse technological and sensory properties such as bitterness and grittiness. Sourdough fermentation is a promising strategy to improve the sensory quality of bran without inducing severe changes to the bran matrix. Therefore, identification of species/strains with potential for industrial sourdough fermentations is important. We compared the effects of different representatives of species of lactic acid bacteria (LAB) on wheat bran. Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus brevis, Lactobacillus sanfranciscensis and Fructobacillus fructosus produced highly individual fermentation patterns as judged from carbohydrate consumption and organic acid production. Interestingly, fructose was released during all bran fermentations and possibly influenced the fermentation profiles of obligately heterofermentative species to varying degrees. Except for the reduction of ferulic acid by L. plantarum and L. pentosus, analyses of phenolic compounds and alkylresorcinols suggested that only minor changes thereof were induced by the LAB metabolism. Sensory analysis of breads baked with fermented bran did not reveal significant differences regarding perceived bitterness and aftertaste. We conclude that in addition to more traditionally used sourdough species such as L. sanfranciscensis and L. brevis, also facultatively heterofermentative species such as L. plantarum and L. pentosus possess potential for industrial wheat bran fermentations and should be considered in further investigations.

Fermented and extruded wheat bran in piglet diets: impact on performance, intestinal morphology, microbial metabolites in chyme and blood lipid radicals.

The aim of the present study was to evaluate the influence of native, fermented and extruded wheat bran on the performance and intestinal morphology of piglets. Additionally, short-chain fatty acids (SCFA), biogenic amines, ammonia, lactic acid, pH as well as E. coli and lactic acid bacterial counts were analysed in digesta samples from three gut sections. Furthermore, the antioxidant potential in blood samples was evaluated based on the lipid radicals formed. For this purpose, 48 newly weaned piglets (28 d old) were allocated to one of the four different dietary treatment groups: no wheat bran (Control), native wheat bran, fermented wheat bran as well as extruded wheat bran. Wheat bran variants were included at 150 g/kg into the diets. All diets were mixed to reach the calculated isonitrogenic nutrient contents. Gut tissue and digesta samples were collected from the proximal jejunum, the terminal ileum and the colon ascendens, blood samples directly at slaughter. Although none of the dietary interventions had an impact on performance parameters, the amount of goblet cells in the ileum was increased upon feeding native and extruded wheat bran, compared to fermented bran (p < 0.05). The E. coli counts in colonic chyme were significantly lower (p < 0.05) in the Control group compared to the groups fed with wheat bran. The concentration of SCFA showed differences for minor compounds (p < 0.05), while linear contrast analyses revealed a reduced concentration of total SCFA in the colon following the feeding of modified wheat bran compared to native wheat bran. This may suggest that several compounds are more easily digested already in the ileum, resulting in a reduced nutrient flow into the large intestine and therefore less unexploited digesta is available as substrate for the microorganisms there. Fermentation also resulted in a significant decrease of methylamine in the colon (p < 0.05), while other biogenic amines in the ileum and colon showed no statistically significant differences. The formation of lipid radicals was decreased (p < 0.05) after feeding native wheat bran compared to the Control group. These results suggest that fermentation and extrusion of wheat bran exert some different impact regarding their physiological mode of action.

Rectal Lactobacillus species and their influence on the vaginal microflora: a model of male-to-female transsexual women.

INTRODUCTION: Based on Lactobacillus species co-colonizing the vagina and rectum, it has been hypothesized that the rectum may be an important reservoir for vaginal colonization by lactobacilli. There are no data on this issue in male-to-female transsexual women. AIM: We undertook this observational study to characterize the Lactobacillus species present in the neovagina and rectum of male-to-female transsexual women and to determine the degree of neovaginal-rectal co-colonization in order to gain a better understanding of the potential role of the gut as a reservoir for genital lactobacilli. METHODS: Sixty-one male-to-female transsexual women with penile skin lined neovagina without clinical signs of infection were recruited on an ongoing basis from among male-to-female transsexual outpatients. Neovaginal and rectal smears were taken for molecular Lactobacillus species profiling by denaturing gradient gel electrophoresis (PCR-DGGE). MAIN OUTCOME MEASURES: Matching Lactobacillus species between neovagina and rectum. RESULTS: Forty-three of the 61 male-to-female transsexual women (70.5%) simultaneously harbored the same lactobacilli in both the neovagina and rectum. We found 276 neovaginal and 258 rectal DGGE bands representing 11 Lactobacillus species, with 201 matches of the same Lactobacillus species in neovagina and rectum. 37 of the 61 women (61%) had two or more matching Lactobacillus species. CONCLUSION: These data support the hypothesis that the rectum may play an important role as source of Lactobacillus species that colonies neovagina of male-to-female transsexual women. In view of the specific anatomical circumstances of the study population, these findings may be extended to the general population of women.

Status quo and future research challenges on organic food quality determination with focus on laboratory methods.

Organic food quality determination needs multi-dimensional evaluation tools. The main focus is on the authentication as an analytical verification of the certification process. New fingerprinting approaches such as ultra-performance liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry, direct analysis in real time-high-resolution mass spectrometry as well as crystallization with and without the presence of additives seem to be promising methods in terms of time of analysis and detecting organic system-related parameters. For further methodological development, a system approach is recommended, which also takes into account food structure aspects. Furthermore, the authentication of processed organic samples needs more consciousness, hence most of organic food is complex and processed.

Arabinoxylan oligosaccharide hydrolysis by family 43 and 51 glycosidases from Lactobacillus brevis DSM 20054.

Due to their potential prebiotic properties, arabinoxylan-derived oligosaccharides [(A)XOS] are of great interest as functional food and feed ingredients. While the (A)XOS metabolism of Bifidobacteriaceae has been extensively studied, information regarding lactic acid bacteria (LAB) is still limited in this context. The aim of the present study was to fill this important gap by characterizing candidate (A)XOS hydrolyzing glycoside hydrolases (GHs) identified in the genome of Lactobacillus brevis DSM 20054. Two putative GH family 43 xylosidases (XynB1 and XynB2) and a GH family 43 arabinofuranosidase (Abf3) were heterologously expressed and characterized. While the function of XynB1 remains unclear, XynB2 could efficiently hydrolyze xylooligosaccharides. Abf3 displayed high specific activity for arabinobiose but could not release arabinose from an (A)XOS preparation. However, two previously reported GH 51 arabinofuranosidases from Lb. brevis were able to specifically remove α-1,3-linked arabinofuranosyl residues from arabino-xylooligosaccharides (AXHm3 specificity). These results imply that Lb. brevis is at least genetically equipped with functional enzymes in order to hydrolyze the depolymerization products of (arabino)xylans and arabinans. The distribution of related genes in Lactobacillales genomes indicates that GH 43 and, especially, GH 51 glycosidase genes are rare among LAB and mainly occur in obligately heterofermentative Lactobacillus spp., Pediococcus spp., members of the Leuconostoc/Weissella branch, and Enterococcus spp. Apart from the prebiotic viewpoint, this information also adds new perspectives on the carbohydrate (i.e., pentose-oligomer) metabolism of LAB species involved in the fermentation of hemicellulose-containing substrates.

The protective effect of farm milk consumption on childhood asthma and atopy: the GABRIELA study.

BACKGROUND: Farm milk consumption has been identified as an exposure that might contribute to the protective effect of farm life on childhood asthma and allergies. The mechanism of action and the role of particular constituents of farm milk, however, are not yet clear. OBJECTIVE: We sought to investigate the farm milk effect and determine responsible milk constituents. METHODS: In rural regions of Germany, Austria, and Switzerland, a comprehensive questionnaire about farm milk consumption and other farm-related exposures was completed by parents of 8334 school-aged children, and 7606 of them provided serum samples to assess specific IgE levels. In 800 cow's milk samples collected at the participants' homes, viable bacterial counts, whey protein levels, and total fat content were analyzed. Asthma, atopy, and hay fever were associated to reported milk consumption and for the first time to objectively measured milk constituents by using multiple regression analyses. RESULTS: Reported raw milk consumption was inversely associated to asthma (adjusted odds ratio [aOR], 0.59; 95% CI, 0.46-0.74), atopy (aOR, 0.74; 95% CI, 0.61-0.90), and hay fever (aOR, 0.51; 95% CI, 0.37-0.69) independent of other farm exposures. Boiled farm milk did not show a protective effect. Total viable bacterial counts and total fat content of milk were not significantly related to asthma or atopy. Increased levels of the whey proteins BSA (aOR for highest vs lowest levels and asthma, 0.53; 95% CI, 0.30-0.97), α-lactalbumin (aOR for interquartile range and asthma, 0.71; 95% CI, 0.52-0.97), and ß-lactoglobulin (aOR for interquartile range and asthma, 0.62; 95% CI, 0.39-0.97), however, were inversely associated with asthma but not with atopy. CONCLUSIONS: The findings suggest that the protective effect of raw milk consumption on asthma might be associated with the whey protein fraction of milk.

Effect of Two Soybean Varieties Treated with Different Heat Intensities on Ileal and Caecal Microbiota in Broiler Chickens.

Soybean products are of high importance for the protein supply of poultry. Heat treatment of soybeans is essential to ensure optimal digestibility because of intrinsic antinutritive factors typical for this feed category. However, excessive treatment promotes the Maillard reaction and reduces protein digestibility. Furthermore, Europe's efforts are to decrease dependence on imports of soybean products and enlarge local production. This process will include an increase in the variability of soybean batches, posing great challenges to adequate processing conditions. Intrinsic soybean properties plus heat treatment intensity might be able to modulate the gut microbiota, which is of crucial importance for an animal's health and performance. To assess the influence of heat treatment and soybean variety on gut microbiota, 2 soybean cakes from 2 varieties were processed at 110 °C or 120 °C and subsequently fed to 336 one-day-old broiler chickens. After 36 days, the animals were slaughtered, and the digesta of the ileum and caecum was collected. Next, 16S rRNA amplicon sequencing of the extracted DNA revealed a high discrepancy between gut sections, but there were no differences between male and female birds. Significant differences attributed to the different soybean varieties and heat intensity were detected for certain bacterial taxa. However, no effect on specific families or genera appeared. In conclusion, the results indicated the potential of processing conditions and soybean variety as microbiota-modulating factors.

Application of plant-based antimicrobials for the growth inhibition of clostridia in pressed beet pulp silage.

BACKGROUND: In this study the inhibition of hop beta acids on the growth of clostridia in soil-contaminated pressed sugar beet pulp silages was investigated. Hop beta acids are natural substances which display their effect at low concentrations. Fresh pressed beet pulp material was mixed with soil to artificially contaminate it with clostridia. Laboratory silos were filled with the substrate, stored at 25 °C and opened for sampling at 0, 2, 8, 15, 30, 60, and 90 days. The impact on clostridial growth during silage fermentation was monitored by determination of the pH value and dry matter content, as well as chemical analysis of the fermentation products. Throughout the experiments, the effect of a commercial silage inoculant based on lactic acid bacteria (LAB) and hop-resistant LAB were examined with and without the combination of plant-based antimicrobials. RESULTS: Results indicate that in contaminated silage samples without any additives high butyric acid contents occurred due to clostridial growth. This spoilage could not be suppressed by the application of LAB, whereas the combined application of LAB and hop beta acids significantly improved silage quality, which was reflected by favourable organic acid composition (P < 0.05). CONCLUSION: The experimental data indicate that the application of hop beta acids improves the preservation effect of LAB in suppressing clostridial growth in silages and thus demonstrates some potential for the combined use of plant-based antimicrobials and LAB.

Interaction of Soybean Varieties and Heat Treatments and Its Effect on Growth Performance and Nutrient Digestibility in Broiler Chickens.

As production of European soybeans is expected to grow, optimal processing conditions need to be ensured for small and heterogeneous batches of soybeans. The effect of different soybean varieties, as well as heat treatments, on the growth performance and nutrient digestibility in broiler chickens was investigated. Two varieties, regarded as heat stable and heat labile after preliminary experiments, were partially de-oiled and thermally processed at 110 °C for 20 min and 120 °C for 20 min. The resulting soybean cakes were integrated into a mash diet and subjected to a 36-day long feeding experiment. A total of 336 one-day-old broiler chickens were divided into 24 pens, resulting in 6 replicates per treatment. With application of the 110 °C treatment, analysis of soybean cakes showed that the commonly required reduction in trypsin inhibitor activity (TIA) was only reached with one soybean variety. The higher processing temperature of 120 °C ensured sufficient TIA reductions in both soybean varieties. Elevated TIA concentrations resulted in decreased growth performances (p < 0.05) of the chickens, whereas no negative effect from overheating on growth performance appeared. Total-tract nitrogen retention (p < 0.05) and pre-caecal digestibility of several amino acids (p < 0.10) decreased with higher processing temperatures but had no negative effects on growth performance. In conclusion, the results indicate that processing conditions adjusted to the different varieties are essential to ensure optimal product quality.

Whole Genome Sequencing-Based Comparison of Food Isolates of Cronobacter sakazakii.

Cronobacter sakazakii is an emerging foodborne pathogen, which is linked to life-threatening infections causing septicemia, meningitis, and necrotizing enterocolitis. These infections have been epidemiologically connected to ingestion of contaminated reconstituted powder infant formula. Even at low water activity C. sakazakii can survive for a long time; it is capable of protective biofilm formation and occasionally shows high virulence and pathogenicity even following stressful environmental conditions. Hence it is a challenging task for the food industry to control contamination of food ingredients and products through the entire production chain, since an increasing number of severe food-related outbreaks of C. sakazakii infections has been observed. The seemingly great capability of C. sakazakii to survive even strict countermeasures combined with its prevalence in many food ingredients requires a greater in depth understanding of its virulence factors to master the food safety issues related to this organism. In this context, we present the whole genome sequence (WGS) of two different C. sakazakii isolated from skimmed milk powder (C7) and ready-to-eat salad mix (C8), respectively. These are compared to other, already sequenced, C. sakazakii genomes. Sequencing of the fusA allele revealed that both isolates were C. sakazakii. We investigated the molecular characteristics of both isolates relevant for genes associated with pathogenesis and virulence factors, resistance to stressful environmental conditions (e.g., osmotic and heat), survival in desiccation as well as conducted a comparative genomic analysis. By using multi-locus sequence typing (MLST), the genetic type of both isolates is assessed and the number of unique genes is determined. DNA of C. sakazakii C8 is shown to hold a novel and unique sequence type; the number of unique genes identified in the genomic sequence of C. sakazakii C7 and C8 were 109 and 188, respectively. Some of the determined unique genes such as the rhs and VgrG genes are linked to the Type VI Secretion System cluster, which is associated with pathogenicity and virulence factors. Moreover, seven genes encoding for multi-drug resistance were found in both isolates. The finding of a number of genes linked to producing capsules and biofilm are likely related to the observed resistance to desiccation.

Characterization of Biofilm Formation by Cronobacter spp. Isolates of Different Food Origin under Model Conditions.

Cronobacter spp. are opportunistic human pathogens that cause serious diseases in neonates and immunocompromised people. Owing to their biofilm formation on various surfaces, both their detection and their removal from production plants constitute a major challenge. In this study, food samples were randomly collected in Austria and examined for the presence of Cronobacter spp. Presumptive isolates were identified by a polyphasic approach. Five percent of the samples were positive for C. sakazakii and 2.4% for C. dublinensis. Individual growth of the isolates was characterized based on lag time, growth rate, and generation time. During an incubation period of 6 to 72 h, biofilm formation of 11 selected isolates was quantified under model conditions by a crystal violet staining assay with 96-well plates with different carbon sources (lactose, glucose, maltose, sucrose, and sodium acetate) and NaCl levels and under variable temperature and pH conditions. Biofilm formation was more pronounced at lactose concentrations between 0.25 and 3% compared with 5% lactose, which lead to thinner layers. C. sakazakii isolate C7, isolated from infant milk powder, was the strongest biofilm producer at 10 mM Mg2+ and 5 mM Mn2+, 0.5% sodium acetate, at pH levels between 7 and 9 at 37°C for 24 h. C. sakazakii strain C6 isolated from a plant air filter was identified as a moderate biofilm former and C. sakazakii strain DSM 4485, a clinical isolate, as a weak biofilm former. Based on PCR detection, genes bcsA, bcsB, and bcsG encoding for cellulose could be identified as markers for biofilm formation. Isolates carrying bcsA and bcsB showed significantly stronger biofilm formation than isolates without these genes ( P < 0.05), in strong correlation with the results obtained in the crystal violet assay. Further investigations using confocal laser scanning microscopy revealed that extracellular polymeric substances and glycocalyx secretions were the dominating components of the biofilms and that the viable fraction of bacteria in the biofilm decreased over time.

Comparison of broth microdilution, Etest, and agar disk diffusion methods for antimicrobial susceptibility testing of Lactobacillus acidophilus group members.

In recent years, the absence of acquired antimicrobial resistance has become an important criterion to evaluate the biosafety of lactobacilli used as industrial starter or probiotic cultures. At present, however, standards for susceptibility testing of Lactobacillus strains or approved guidelines for interpreting the test results are not available. Hence, this study was carried out to contribute to the establishment of a standardized procedure for antimicrobial susceptibility testing of lactobacilli. The results obtained by testing 104 strains of the Lactobacillus acidophilus group were compared based on broth microdilution, disk diffusion, and Etest. Except for some specific agent-related effects, agreement between MICs resulting from the broth microdilution method and the Etest was good. In addition, inhibition zone diameters determined with disk diffusion correlated well with MICs from Etest and broth microdilution.