Short communication: Clostridial spore counts in vat milk of Alpine dairies.

One of the most severe quality defects in hard and semi-hard cheese, the late blowing defect, is caused by endospore-forming bacteria of the genus Clostridium. To minimize financial losses and waste of resources due to cheese spoilage, raw milk with elevated clostridial spore counts should not be used for the production of certain cheese types. In this context, threshold values of clostridial spore concentrations that cause quality defects in cheese are still under debate. To improve our understanding about late blowing defects, further information on the correlation between clostridial spore concentrations in milk and cheese quality is indispensable. Thus, the aim of this study was to monitor the microbiological quality of milk used for Alpine cheese production regarding clostridial endospore levels to facilitate the establishment of threshold spore concentrations that guarantee the absence of quality defects in Austrian cheese. For this purpose, we monitored clostridial endospore levels in vat milk of 4 Alpine dairies throughout the summer grazing period in 2018. Surprisingly, we observed almost complete absence of butyric acid-producing clostridia in milk and no blowing defects in cheese. Hence, critical clostridial spore concentrations could not be verified. Moreover, the observed low spore levels reveal that the prohibition of silage feeding and good farming practices effectively minimize clostridial endospore counts in milk and ensure the manufacture of high-quality cheese even if technological possibilities are limited.

A diet containing native or fermented wheat bran does not interfere with natural microbiota of laying hens.

Wheat bran (WB) is an important side product of the milling industry and can serve as dietary fiber compound for monogastric animals. The aim of this study was to evaluate the influence of native or fermented WB on the gut physiology and microbiology of laying hens. To accomplish this, 24 laying hens were fed the following diets: conventional diet without WB; 15% native WB in the diet; 15% WB fermented with Pleurotus eryngii; and 15% WB fermented with P. eryngii and a lactic acid bacterial culture. Immediately after slaughtering, digesta samples were taken from the jejunum, ileum and cecum, respectively. Total DNA was extracted and subsequently investigated with 16S DNA amplicon sequencing. Neither native nor fermented WB supplementations negatively affected the feed conversion ratio, laying performance or the relative abundances and alpha-diversity of microbiota in the intestine. Effects of WB-based diets on gut morphology were only recognized in the jejunum (reduced villum height and mucosa thickness). Likewise, WB supplementation decreased the digestibility of DM and starch. Based on these findings, it was demonstrated that different WB variants are applicable without exerting practically negative consequences on performance or on gut microbiota. Fermentation improved the digestibility/retention of dietary fat and phosphorus. However, no further beneficial effects were observed. This study also allowed a more in-depth view on the laying hens' gut microbiome and its variation within the gut segments.

Effect of a yoghurt drink containing Lactobacillus strains on bacterial vaginosis in women - a double-blind, randomised, controlled clinical pilot trial.

Bacterial vaginosis (BV) is characterised by a depletion of lactobacilli in favour of an overgrowth of anaerobic bacteria. It is associated with increased risk for urogenital infections and abortion. In this study we assessed the effect of a yoghurt drink containing Lactobacillus strains on BV. The strains had been isolated from healthy pregnant women and selected for acidification capacity, production of H2O2, glycogen utilisation, bile salt tolerance and inhibition of pathogens. Using Amsel criteria BV was diagnosed in 36 women aged ≥18 years with stable menstrual cycle or menopause. They were treated with oral metronidazole for 7 days (2×500 mg/d). Starting with the treatment, women consumed twice daily either verum or placebo during 4 weeks. Verum was 125 g yoghurt containing (besides Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus) living strains Lactobacillus crispatus LbV 88 (DSM 22566), Lactobacillus gasseri LbV 150N (DSM 22583), Lactobacillus jensenii LbV 116 (DSM 22567) and Lactobacillus rhamnosus LbV96 (DSM 22560), each 1×107 cfu/ml; placebo was 125 g chemically acidified milk. After 4 weeks of intervention 0 of 17 had BV in the verum group versus 6 of 17 in the s.a. control (0.018 in Fisher Exact test). Amsel score decreased during the intervention period by 4.0 (median) (4.0; 3.0) (25th; 75th percentile) in the verum group compared to 2.0 (4.0; 0.0) in the control group (P=0.038 in Mann-Whitney test). Discharge and odour (Amsel criteria 2+3) also decreased by 2.0 (2.0; 1.0) in the verum compared to 1.0 (2.0; 0.0) in the control group (P=0.01) and differed after 4 weeks intervention between the groups 0.0 (0.0; 0.0) versus 1.0 (0.0; 2.0) (P=0.001). Nugent score decreased during the intervention period by 5.5 (7.0;2.3) in the verum compared to 3.0 (6.0;0.5) in the control group (P=0.158). Additional intake of yoghurt containing these probiotic strains improved the recovery rate and symptoms of BV and tended to improve the vaginal microbial pattern.

Akkermansia muciniphila: a novel functional microbe with probiotic properties.

Akkermansia muciniphila is an intestinal anaerobe which has been proposed as a new functional microbe with probiotic properties. However, the species is not included in the European Union qualified presumption of safety (QPS) list and has not yet been assessed. Moreover, products containing A. muciniphila are not on the market and are thus controlled by the Novel Foods Regulation, which requires extensive safety assessment. This review addresses the safety aspects of the use of A. muciniphila based on published information on its functions in humans and predictions based on its activity in model animals. Further, comprehensive studies related to A. muciniphila and its safety properties have gradually appeared and are summarised here. Many of the criteria required for novel food safety assessment in Europe can thus be fulfilled. However, studies focusing on the toxicological properties of A. muciniphila, including long-term and reproduction studies, have not so far been reported and are discussed in the light of the observation that most, if not all, healthy subjects are known to carry this intestinal anaerobe. As this also applies to other beneficial bacteria found in the human intestinal tract, the A. muciniphila case can be seen as a model for the comprehensive safety evaluations required by the European authorities.

Pulsed Light Treatment of Different Food Types with a Special Focus on Meat: A Critical Review.

Today, the increasing demand for minimally processed foods that are at the same moment nutritious, organoleptically satisfactory, and free from microbial hazards challenges the research and development to establish alternative methods to reduce the level of bacterial contamination. As one of the recent emerging nonthermal methods, pulsed light (PL) constitutes a technology for the fast, mild, and residue-free surface decontamination of food and food contact materials in the processing environment. Via high frequency, high intensity pulses of broad-spectrum light rich in the UV fraction, viable cells as well as spores are inactivated in a nonselective multi-target process that rapidly overwhelms cell functions and subsequently leads to cell death. This review provides specific information on the technology of pulsed light and its suitability for unpackaged and packaged meat and meat products as well as food contact materials like production surfaces, cutting tools, and packaging materials. The advantages, limitations, risks, and essential process criteria to work efficiently are illustrated and discussed with relation to implementation on industrial level and future aspects. Other issues addressed by this paper are the need to take care of the associated parameters such as alteration of the product and utilized packaging material to satisfy consumers and other stakeholders.

Strategies for the evaluation and selection of potential vaginal probiotics from human sources: an exemplary study.

During the last years, the application of probiotics in gynaecological clinical practice has gained increasing relevance regarding therapy and prevention. This trend has also provoked the need for having tailored pharmaceutical preparations containing powerful microbial strains with defined properties. For the development of such preparations, several factors and criteria have to be considered, thereby not only focusing on identity and safety aspects as well as individual properties of the bacterial strains, but also on technological issues, such as stability and targeted release from the preparation. Against this background, this report exemplarily addresses the development procedure of a probiotic bacterial formulation for gynaecological application, covering the search for suitable strains, assessing their microbiological, molecular biological and physiological characterisation, and the selection for their use in clinical trials. In detail, starting with 127 presumptive lactobacilli isolates of vaginal origin, a step-by-step selection of candidate strains meeting special criteria was thoroughly examined, finally leading to a preparation consisting of four individual Lactobacillus strains that possess particular significance in women's urogenital health. Relevant issues and quality criteria of probiotic preparations used in gynecology are addressed and exemplarily introduced.

Differences in the vaginal lactobacilli of postmenopausal women and influence of rectal lactobacilli.

OBJECTIVE: This study was undertaken to characterize the Lactobacillus spp. dominating the vaginal microbiota of healthy postmenopausal women and to determine the possible influence of rectal lactobacilli. METHODS: Sixty postmenopausal women aged 55-65 years without clinical signs of vaginal infection not receiving hormone replacement therapy were included in this cross-sectional observational study. Based on Gram-stained smears, 30 women with a normal vaginal flora (Nugent score 0) were included in Group 1, and 30 women with an intermediate vaginal flora characterized by an absence of vaginal lactobacilli (Nugent score 4) were included in Group 2. Vaginal and rectal smears were taken for molecular lactobacillus profiling using polymerase chain reaction and denaturing gradient gel electrophoresis. Diversity of vaginal and rectal lactobacilli in postmenopausal women was the main outcome measure. RESULTS: We noticed a minor interference of gut lactic acid bacteria on a normal vaginal microflora dominated by lactobacilli strains of the L. delbrueckii group. When the normal vaginal microflora is disturbed by depletion of lactobacilli, the gut may function as a reservoir for lactobacilli of the L. casei group, which then colonize the vagina. CONCLUSION: Our data indicate that rectal lactobacilli may affect the vaginal flora of postmenopausal women in the case of lactobacillary absence and help to maintain a normal vaginal microbiota.

Short communication: appropriate and alternative methods to determine viable bacterial counts in cow milk samples.

Farm milk consumption is reported to be inversely related to the development of asthma and atopy in children and it has been hypothesized that microorganisms in milk might contribute to this protective effect. The GABRIEL study was designed to investigate this hypothesis in a large population of European children, calling for a rapid alternative to classical culture techniques to determine bacteriological properties of milk samples. One objective was to evaluate 2 different rapid methods to determine bacteriological properties in a large number of cow milk samples collected under field conditions. BactoScan (Foss Analytical, Hillerød, Denmark), an automated standard flow cytometric method utilized for routine testing of milk quality, and TEMPO (bioMérieux, Marcy l'Etoile, France), an automated most-probable-number method, were used to assess the total viable bacterial count in farm and commercial milk samples. Both methods were compared with standard plate count method and each other. Measurements based on the TEMPO method were in good agreement with the standard plate count method and showed reliable results, whereas BactoScan results did not correlate with standard plate count measurements and yielded higher bacteria counts in heat-treated milk samples compared with raw milk samples. Most likely, these discrepant results were due to inferences with staining reactions and detection of bacteria in heat-treated milk samples. We conclude that, in contrast to the routinely used BactoScan method, the TEMPO method is an inexpensive and rapid alternative to standard culture methods suitable to assess total bacterial counts in processed and raw milk samples.

Monitoring transmission routes of Listeria spp. in smoked salmon production with repetitive element sequence-based PCR techniques.

Various techniques have been used for tracing the transmission routes of Listeria species and for the assessment of hygiene standards in food processing plants. The potential of repetitive element sequence-based PCR (Rep-PCR) methods (GTG5 and REPI + II) for the typing of Listeria isolates (n = 116), including Listeria monocytogenes (n = 46), was evaluated in a particular situation arising from the relocation of a company producing cold-smoked salmon. Pulsed-field gel electrophoresis (PFGE) using three restriction enzymes (ApaI, AscI, and SmaI) was used for comparison. Identical transmission scenarios among two companies could be identified by cluster analysis of L. monocytogenes isolates that were indistinguishable by both Rep-PCR and PFGE. The calculated diversity index (DI) indicates that Rep-PCR subtyping of Listeria species with primer sets GTG5 and REPI + II has a lower discrimination power than does PFGE. When concatenated Rep-PCR cluster analysis was used, the DI increased from 0.934 (REPI + II) and 0.923 (GTG5) to 0.956. The discrimination power of this method was similar to that of PFGE typing based on restriction enzyme Apa I (DI = 0.955). Listeria welshimeri may be useful as an indicator for monitoring smoked salmon processing environments. Rep-PCR meets the expectations of a reasonable, fast, and low-cost molecular subtyping method for the routine monitoring of Listeria species. The discriminatory power as characterized by the DI sufficiently quantifies the probability of unrelated isolates being characterized as different subtypes. Therefore, Rep-PCR typing based on two primer systems (GTG5 and REPI + II) may be a useful tool for monitoring industrial hygiene.

Vaginal Lactobacillus microbiota of healthy women in the late first trimester of pregnancy.

OBJECTIVE: This study was undertaken to characterise the dominant species of Lactobacillus colonising the vagina of healthy pregnant women, to examine some of their phenotypic and genotypic properties, and to gain a better understanding of the potential role of species, which might be associated with infection-free status. DESIGN: A prospective descriptive cohort study. SETTING: Department of Obstetrics and Gynaecology, Medical University of Vienna and Medical School, Vienna, Austria. SAMPLE: A total of 200 women in the late first trimester of pregnancy without clinical signs of vaginal infection were included in the study. Of these, 126 women were found to have a normal vaginal flora based on Gram stain. METHODS: Culture probes from those 126 women were further processed for identification of Lactobacillus species. Overall, 168 colonies from 84 women were identified as belonging to the Lactobacillus genus. Based on the combined results of microbiological methods and genus-specific, multiplex, and species-specific polymerase chain reaction, lactobacilli were recovered from 72 women. MAIN OUTCOME MEASURES: Identification of Lactobacillus species of the vaginal flora of healthy pregnant women. RESULTS: The most frequently occurring species were Lactobacillus crispatus and Lactobacillus gasseri, followed by Lactobacillus jensenii and Lactobacillus rhamnosus. CONCLUSIONS: Our results may have implications on the composition and on the use of Lactobacillus preparations for the prevention of recurrent vaginal infection.

Enumeration of probiotic bacilli spores in animal feed: interlaboratory study.

Fourteen out of 17 laboratories completed an interlaboratory study comparing 2 pretreatment protocols of feed samples containing authorized probiotic bacilli spores. Both methods used tryptone soy agar for enumeration. Pretreatment A involved preparation of a suspension of the feed sample in 50% ethanol. For pretreatment B, the sample was suspended in peptone salt solution and heated at 80 degrees C for 10 min. Each laboratory analyzed 12 samples (6 per pretreatment), which represented duplicates of a high (10(9) colony-forming units [CFU]/g) and low (10(5) CFU/g) level of bacilli spores or a blank that contained vegetative probiotic bacteria only. For pretreatment A, the repeatability relative standard deviation (RSD(r)) was 2.9% for the low level and 2.5% for the high. The reproducibility relative standard deviation (RSDR) values were 7.8 and 5.9%, respectively. Pretreatment B revealed RSD(r) values of 1.1 and 1.0%, and RSDR values of 5.8 and 3.4%, respectively. The heat treatment (pretreatment B) of feed samples had better precision data, resulted in higher viable bacilli counts, and was more effective in deactivating vegetative background flora. It is therefore recommended for adoption for official control purposes and for CEN and ISO standards.

A collaborative study of a method for enumeration of probiotic enterococci in animal feed.

AIMS: Validation of an enumeration method to be used as an official control method in the framework of Council Directive 70/524/EEC for probiotic enterococci used as feed additives. METHODS AND RESULTS: Twenty laboratories in 12 European countries carried out a collaborative study. A plate count method using bile esculin azide (BEA) agar was used. Precision data in terms of repeatability (r) and reproducibility (R) of the method using different feeding stuffs and three inoculation levels were determined. Enterococci were present in the samples as a single component or in mixtures with other probiotic feed additives. The enumeration of enterococci on BEA agar showed a relative standard deviation (RSD)r of 1.5-3.6% and an RSD(R) between 2.9 and 7.4%. BEA agar was selective for enterococci in the presence of other probiotic micro-organisms such as pediococci, lactobacilli and yeast. CONCLUSIONS: For routine analysis of viable enterococci concentrations in feeding stuffs, the use of BEA is recommended. This methodology is not applicable for mineral feeds. SIGNIFICANCE AND IMPACT OF THE STUDY: An official control method for enumeration of authorized probiotic enterococci in feeding stuffs was validated. The results are intended for consideration for adoption as CEN and ISO standards.

Clustering of Saccharomyces boulardii strains within the species S. cerevisiae using molecular typing techniques.

AIMS: This study was undertaken to characterize and differentiate therapeutically relevant Saccharomyces yeasts. Among the isolates were so-called Saccharomyces boulardii strains, which are considered as probiotic agents, but whose taxonomic assignment is controversial. Moreover, the discriminative power of the applied molecular typing techniques should be evaluated. METHODS AND RESULTS: Genotyping was performed using species-specific polymerase chain reaction (PCR), randomly amplified polymorphic DNA-PCR, restriction fragment length polymorphism analysis of rDNA spacer regions and pulsed-field gel electrophoresis. Species-specific PCR assigned all of the product isolates to the species S. cerevisiae. By combining the other techniques, all isolates could be discriminated. Moreover, it could be demonstrated that probiotic S. boulardii strains form a separate cluster located within the species. CONCLUSIONS: With the exception of species-specific PCR, all of the applied methodologies were suitable for subspecies typing and indicated a close relationship between the probiotic strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods applied in this study are considered powerful tools for quality control of therapeutically relevant yeasts. It is of crucial importance, especially regarding S. boulardii yeasts, to verify the identity of the correct strain, since the beneficial properties are considered to be strain-specific.

Endocarditis by Lactobacillus rhamnosus due to yogurt ingestion?

A young man who ate large quantities of probiotic yogurt developed endocarditis and septic arthritis caused by Lactobacillus rhamnosus. The pathogenic isolate could not be distinguished from the yogurt microflora using methods routinely used in the clinical microbiology laboratory. Only by using more appropriate methodology, including PCR, the pathogen could be distinguished from the yogurt isolate.

Utilization of prebiotic carbohydrates by yeasts of therapeutic relevance.

AIMS: To investigate 17 strains of therapeutically relevant strains of Saccharomyces cerevisiae (including 10 strains of so-called S. boulardii) isolated from various pharmaceutical products, feed supplements and brewer's yeast for their capability of utilizing selected carbohydrates of prebiotic importance. METHODS AND RESULTS: Automated turbidimetric measurements and conventional test combinations were used to examine the basic sugar assimilation profiles of the test strains. It was shown that none of the so-called S. boulardii strains utilized galactose and palatinose. Among the prebiotic substrates, the yeasts indicated a pronounced preference for metabolizing the fructo-oligosaccharides. CONCLUSION: Yeast strains of therapeutic relevance can be successfully combined with certain prebiotics in symbiotic formulations. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study may serve as a basis for the development of new pharmaceutical preparations for medical therapy and a better understanding of intestinal micro-ecology.

Continuous venovenous hemofiltration improves arterial oxygenation in endotoxin-induced lung injury in pigs.

BACKGROUND: Hypoxemia is common in septic acute lung failure. Therapy is mainly supportive, and most trials using specific inhibitors of key inflammatory mediators (ie., tumor necrosis factor alpha, interleukin 1) have failed to prove beneficial. The authors investigated if a nonspecific blood purification technique, using zero-balanced high-volume continuous venovenous hemofiltration (CWH), might improve arterial oxygenation in a fluid-resuscitated porcine model of endotoxin-induced acute lung injury. METHODS: Piglets of both sexes weighing 25-30 kg were anesthetized and mechanically ventilated. After baseline measurements, animals received an intravenous infusion of 0.5 mg/kg endotoxin (Escherichia coli lipopolysaccharide). One hour after endotoxin, animals were randomly assigned to either treatment with CWH (endotoxin + hemofiltration, n = 6) or spontaneous course (endotoxin, n = 6). At 4 h after randomization, animals were killed. Hemofiltration was performed from femoral vein to femoral vein using a standard circuit with an EF60 polysulphone hemofilter. RESULTS: Endotoxin challenge induced arterial hypoxemia, an increase in peak inspiratory pressure, pulmonary hypertension, and systemic hypotension. Treatment with CWH did not improve systemic or pulmonary hemodynamics. However, arterial oxygenation was increased in endotoxin-challenged animals at 5 h after completion of endotoxin infusion, as compared with animals not receiving CVVH (arterialoxygen tension, 268+/-33 vs. 176+/-67 mm/Hg, respectively, P < 0.01). In addition, treatment with CWH attenuated the endotoxin-induced increase in peak inspiratory pressure and increased lung compliance. CONCLUSION: These results suggest that nonspecific blood purification with high-volume CWH improves arterial oxygenation and lung function in endotoxin-induced acute lung injury in pigs, independent of improved hemodynamics, fluid removal, or body temperature.

Microbiological status of commercially available medicinal herbal drugs--a screening study.

One hundred and thirty-eight medicinal herbal drugs obtained from different suppliers were examined for microbial contaminants and for the detectability of pathogenic microorganisms. For this purpose, several microbiological standard parameters (total aerobic mesophilic count, enterobacteria, coliforms, aerobic sporeformers, yeasts and moulds, enterococci, lactobacilli, pseudomonades and aeromades) and selective methods for the detection of indicator microorganisms and pathogens (E. coli, enterohaemorrhagic E. coli [EHEC], Salmonella, Campylobacter jejuni, Pseudomonas aeruginosa, Bacillus cereus, Clostridium perfringens, Listeria, coagulase-positive staphylococci, Candida albicans, potentially aflatoxigenic moulds) were applied. The microbial load of the samples varied considerably. While none of the samples contained EHEC, Salmonellae, Pseudomonas aeruginosa, Listeriae, Staphylococcus aureus or Candida albicans, four samples were E. coli positive, two samples were presumptively Campylobacter jejuni positive and nine herbal drugs contained a potentially aflatoxigenic mould flora. Further details regarding different viable count classes as well as preparation techniques are discussed.

Influence of a synbiotic mixture consisting of Lactobacillus acidophilus 74-2 and a fructooligosaccharide preparation on the microbial ecology sustained in a simulation of the human intestinal microbial ecosystem (SHIME reactor).

Lactobacillus acidophilus 74-2, which is used in probiotic products, was administered, with fructo-oligosaccharide in a milk-based product, to the second vessel (duodenum/jejunum) of the SHIME reactor, an in vitro simulation of the human intestinal microbial ecology. The main focus of this study was to monitor the changes of the population density of selected bacterial species in the intestine and the changes of metabolic activities during the supplementation of L. acidophilus and fructooligosaccharide in the SHIME reactor. Interestingly, the addition of L. acidophilus 74-2 with fructooligosaccharide gave rise to an increase of bifidobacteria. Moreover, major positive changes occurred in the production of volatile fatty acids: a strong upward trend was observed especially in the case of butyric acid and propionic acid. Furthermore a noticeable increase of beta-galactosidase activity was monitored, while the activity of beta-glucuronidase, generally considered undesirable, declined.