Abundance and potential contribution of Gram-negative cheese rind bacteria from Austrian artisanal hard cheeses.

Many different Gram-negative bacteria have been shown to be present on cheese rinds. Their contribution to cheese ripening is however, only partially understood until now. Here, cheese rind samples were taken from Vorarlberger Bergkäse (VB), an artisanal hard washed-rind cheese from Austria. Ripening cellars of two cheese production facilities in Austria were sampled at the day of production and after 14, 30, 90 and 160days of ripening. To obtain insights into the possible contribution of Advenella, Psychrobacter, and Psychroflexus to cheese ripening, we sequenced and analyzed the genomes of one strain of each genus isolated from VB cheese rinds. Additionally, quantitative PCRs (qPCRs) were performed to follow the abundance of Advenella, Psychrobacter, and Psychroflexus on VB rinds during ripening in both facilities. qPCR results showed that Psychrobacter was most abundant on cheese rinds and the abundance of Advenella decreased throughout the first month of ripening and increased significantly after 30days of ripening (p<0.01). Psychrobacter and Psychroflexus increased significantly during the first 30 ripening days (p<0.01), and decreased to their initial abundance during the rest of the ripening time (p<0.05). Genome sequencing resulted in 17 to 27 contigs with assembly sizes of 2.7 Mbp for Psychroflexus, 3 Mbp for Psychrobacter, and 4.3 Mbp for Advenella. Our results reveal that each genome harbors enzymes shown to be important for cheese ripening in other bacteria such as: Cystathionine/Methionine beta or gamma-lyases, many proteases and peptidases (including proline iminopeptidases), aminotransferases, and lipases. Thus, all three isolates have the potential to contribute positively to cheese ripening. In conclusion, the three species quantified were stable community members throughout the ripening process and their abundance on cheese rinds together with the results from genome sequencing suggest an important contribution of these bacteria to cheese ripening.

Bacterial diversity of floor drain biofilms and drain waters in a Listeria monocytogenes contaminated food processing environment.

Sanitation protocols are applied on a daily basis in food processing facilities to prevent the risk of cross-contamination with spoilage organisms. Floor drain water serves along with product-associated samples (slicer dust, brine or cheese smear) as an important hygiene indicator in monitoring Listeria monocytogenes in food processing facilities. Microbial communities of floor drains are representative for each processing area and are influenced to a large degree by food residues, liquid effluents and washing water. The microbial communities of drain water are steadily changing, whereas drain biofilms provide more stable niches. Bacterial communities of four floor drains were characterized using 16S rRNA gene pyrosequencing to better understand the composition and exchange of drain water and drain biofilm communities. Furthermore, the L. monocytogenes contamination status of each floor drain was determined by applying cultivation-independent real-time PCR quantification and cultivation-dependent detection according to ISO11290-1. Pyrosequencing of 16S rRNA genes of drain water and drain biofilm bacterial communities yielded 50,611 reads, which were clustered into 641 operational taxonomic units (OTUs), affiliated to 16 phyla dominated by Proteobacteria, Firmicutes and Bacteroidetes. The most abundant OTUs represented either product- (Lactococcus lactis) or fermentation- and food spoilage-associated phylotypes (Pseudomonas mucidolens, Pseudomonas fragi, Leuconostoc citreum, and Acetobacter tropicalis). The microbial communities in DW and DB samples were distinct in each sample type and throughout the whole processing plant, indicating the presence of indigenous specific microbial communities in each processing compartment. The microbiota of drain biofilms was largely different from the microbiota of the drain water. A sampling approach based on drain water alone may thus only provide reliable information on planktonic bacterial cells but might not allow conclusions on the bacterial composition of the microbiota in biofilms.

Pyrosequencing reveals diverse fecal microbiota in Simmental calves during early development.

From birth to the time after weaning the gastrointestinal microbiota of calves must develop into a stable, autochthonous community accompanied by pivotal changes of anatomy and physiology of the gastrointestinal tract. The aim of this pilot study was to examine the fecal microbiota of six Simmental dairy calves to investigate time-dependent dynamics of the microbial community. Calves were followed up from birth until after weaning according to characteristic timepoints during physiological development of the gastrointestinal tract. Pyrosequencing of 16S rRNA gene amplicons from 35 samples yielded 253,528 reads clustering into 5410 operational taxonomic units based on 0.03 16S rRNA distance. Operational taxonomic units were assigned to 296 genera and 17 phyla with Bacteroidetes, Firmicutes, and Proteobacteria being most abundant. An age-dependent increasing diversity and species richness was observed. Highest similarities between fecal microbial communities were found around weaning compared with timepoints from birth to the middle of the milk feeding period. Principal coordinate analysis revealed a high variance particularly in samples taken at the middle of the milk feeding period (at the age of approximately 40 days) compared to earlier timepoints, confirming a unique individual development of the fecal microbiota of each calf. This study provides first deep insights into the composition of the fecal microbiota of Simmental dairy calves and might be a basis for future more detailed studies.

Cultivation-independent analysis of microbial communities on Austrian raw milk hard cheese rinds.

"Vorarlberger Bergkäse" (VB) is an Austrian artisanal hard cheese produced from raw cow's milk. The composition of its rind microbiota and the changes in the microbial communities during ripening have not previously been investigated. This study used 16S and 18S rRNA gene cloning and sequencing to characterize the bacterial and fungal communities of seven pooled cheese rind samples taken in seven different ripening cellars of three Austrian dairy facilities. A total of 408 clones for 16S and 322 clones for 18S rRNA gene libraries were used for taxonomic classification, revealing 39 bacterial and seven fungal operational taxonomic units (OTUs). Bacterial OTUs belonged to four different phyla. Most OTUs were affiliated to genera often found in cheese, including high numbers of coryneforms. The most abundant OTU from 16S rRNA gene libraries showed highest similarity to Halomonas. Young cheese rinds were dominated by Actinobacteria or Proteobacteria, particularly by Halomonas and Brevibacterium aurantiacum, while Staphyloccocus equorum was most abundant in old cheeses. The most abundant 18S rRNA OTU had highest similarity to the filamentous fungus Scopulariopsis brevicaulis. Pairwise correlation analyses revealed putative co-occurrences between a number of OTUs. It was possible to discriminate the different cheese rind microbiota at the community-level by facility affiliation and ripening time. This work provides insights into the microbial composition of VB cheese rinds and might allow the processing- and ripening conditions to be improved to enhance the quality of the product.

Metabolic and hormonal responses to subcutaneous glucagon in healthy beagles.

OBJECTIVE: To compare the effects of subcutaneous (SC) and intravenous (IV) glucagon on glucose concentrations, and insulin and cortisol secretion. DESIGN: Prospective randomized 3-way crossover study. SETTING: University teaching hospital. ANIMALS: Five healthy beagles. INTERVENTIONS: Diabetes mellitus and adrenal insufficiency were excluded by repeated glucose and fructosamine measurements, urinalysis, abdominal ultrasonography, and ACTH stimulation tests. Blood samples were collected before and after the SC and IV injection of 1 milligram (1 mg = 1 mL) commercially available synthetic glucagon and analyzed for insulin-like immunoreactivity (insulin-imr), glucose, ACTH and cortisol concentrations. The results were compared with those obtained after the SC injection of 1 mL saline (placebo). Measurements were performed over a period of up to 3 hours. MEASUREMENTS AND MAIN RESULTS: SC glucagon significantly increased glucose and insulin-imr (P < 0.001 and 0.043, respectively). Peak glucose concentrations were observed after 20 minutes and were lower than after IV injection (mean ± SD: 6.5 ± 1.1 mmol/L versus 9.3 ± 0.8 mmol/L [117.1 ± 19.8 mg/dL versus 167.6 ± 14.4 mg/dL]; P = 0.001). The route of application had no significant effect on insulin-imr (peak concentration: median [range]: 83.3 [13.9-312.5] pmol/L versus 194.5 [118.1-284.7] pmol/L [12 [2-45] µU/mL versus 28 [17-41] µU/mL; P = 0.151). SC glucagon did not increase cortisol or ACTH concentrations at any time point of observation (P > 0.05). Aside from somnolence, no adverse events were recorded. CONCLUSIONS: SC glucagon has the potential to be used as a simple and safe test in diabetic animals, but is of little use in animals with suspected corticotrophic insufficiency. The hyperglycemic effects are significant, implying that the commercially available human emergency kit could be useful in the home treatment of canine hypoglycemic emergencies.