Facklamia lactis sp. nov., isolated from raw milk.

Two strains of a Gram-staining-positive species were isolated from German bulk tank milk. On the basis of their 16S rRNA sequences they were affiliated to the genus Facklamia but could not be assigned to any species with a validly published name. Facklamia miroungae ATCC BAA-466T (97.3 % 16S rRNA sequence similarity), Facklamia languida CCUG 37842T (96.9 %), and Facklamia hominis CCUG 36813T (96.6 %) are the closest relatives. In the 16S rRNA phylogeny and in the core-genome phylogeny strains WS 5301T and WS 5302 form a well-supported, separate lineage. Pairwise average nucleotide identity calculated using MUMmer (ANIm) between WS 5301T and type strains of other Facklamia species is well below the species cut-off (95 %) and ranges from 83.4 to 87.7 %. The DNA G+C content of the type strain is 36.4 mol% and the assembly size of the genome is 2.2 Mb. Cells of WS 5301T are non-motile, non-endospore-forming, oxidase-negative, catalase-negative and facultatively anaerobic cocci. The fastidious species grows at 10-40 °C and with up to 7.0 % (w/v) NaCl in BHI supplemented with 5 g l-1 yeast extract. Major polar lipids are phosphatidylglycerol, diphosphatidylglycerol and two glycolipids. Predominant fatty acids are C16 : 1ω9c and C18 : 1ω9c. On the basis of their genomic, physiological and chemotaxonomic characteristics the strains examined in this study represent the same, hitherto unknown species. We propose the name Facklamia lactis sp. nov. for which WS 5301T (=DSM 111018T=LMG 31861T) is the type strain and WS 5302 (=DSM 111019=LMG 31862) is an additional strain of this novel species.

Amplicon-sequencing of raw milk microbiota: impact of DNA extraction and library-PCR.

The highly complex raw milk matrix challenges the sample preparation for amplicon-sequencing due to low bacterial counts and high amounts of eukaryotic DNA originating from the cow. In this study, we optimized the extraction of bacterial DNA from raw milk for microbiome analysis and evaluated the impact of cycle numbers in the library-PCR. The selective lysis of eukaryotic cells by proteinase K and digestion of released DNA before bacterial lysis resulted in a high reduction of mostly eukaryotic DNA and increased the proportion of bacterial DNA. Comparative microbiome analysis showed that a combined enzymatic and mechanical lysis procedure using the DNeasy® PowerFood® Microbial Kit with a modified protocol was best suitable to achieve high DNA quantities after library-PCR and broad coverage of detected bacterial biodiversity. Increasing cycle numbers during library-PCR systematically altered results for species and beta-diversity with a tendency to overrepresentation or underrepresentation of particular taxa. To limit PCR bias, high cycle numbers should thus be avoided. An optimized DNA extraction yielding sufficient bacterial DNA and enabling higher PCR efficiency is fundamental for successful library preparation. We suggest that a protocol using ethylenediaminetetraacetic acid (EDTA) to resolve casein micelles, selective lysis of somatic cells, extraction of bacterial DNA with a combination of mechanical and enzymatic lysis, and restriction of PCR cycles for analysis of raw milk microbiomes is optimal even for samples with low bacterial numbers. KEY POINTS: • Sample preparation for high-throughput 16S rRNA gene sequencing of raw milk microbiota. • Reduction of eukaryotic DNA by enzymatic digestion. • Shift of detected microbiome caused by high cycle numbers in library-PCR.

Pseudomonas cremoris sp. nov., a novel proteolytic species isolated from cream.

During a study investigating the microbiota of raw milk and its semi-finished products, strains WS 5106T and WS 5096 were isolated from cream and skimmed milk concentrate. They could be assigned to the genus Pseudomonas by their 16S rRNA sequences, but not to any validly named species. In this work, a polyphasic approach was used to characterize the novel strains and to investigate their taxonomic status. Examinations based on the topology of core genome phylogenomy as well as average nucleotide identity (ANIm) comparisons suggested a novel Pseudomonas species within the Pseudomonas fluorescens subgroup. With pairwise ANIm values of 90.1 and 89.8 %, WS 5106T was most closely related to Pseudomonas nabeulensis CECT 9765T and Pseudomonas kairouanensis CECT 9766T. The G+C content of strain WS 5106T was 60.1 mol%. Morphologic analyses revealed Gram-stain-negative, aerobic, catalase and oxidase positive, rod-shaped and motile cells. Proteolysis on skimmed milk agar as well as lipolysis on tributyrin agar occurred at both 28 and 6 °C. Tolerated growth conditions were temperatures between 4 and 34 °C, pH values between 6.0 and 8.0, and salt concentrations of up to 5 %. Fatty acid profiles showed a pattern typical for Pseudomonas, with C16 : 0 as the dominant component. The major cellular polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol and the dominating quinone was Q-9. Based on these results, it is proposed to classify the strains as a novel species, Pseudomonas cremoris sp. nov., with WS 5106T (=DSM 111143T=LMG 31863T) as type strain and WS 5096 (=DSM 111129=LMG 31864) as an additional strain.

Complementary Use of Cultivation and High-Throughput Amplicon Sequencing Reveals High Biodiversity Within Raw Milk Microbiota.

Raw milk microbiota are complex communities with a significant impact on the hygienic, sensory and technological quality of milk products. However, there is a lack of knowledge on factors determining their composition. In the present study, four bulk tank milk samples of two farms at two different time points were analyzed in detail for their microbiota using cultivation and 16S rRNA amplicon sequencing. Diversity in samples from the first time point was assessed via cultivation of 500 aerobic mesophilic bacterial isolates in each sample. A high biodiversity of 70 and 110 species per sample was determined, of which 25-28% corresponded to yet unknown taxa. The isolates were dominated by Gram-positive members of the genera Staphylococcus, Corynebacterium, Streptococcus, or Janibacter, whilst Chryseobacterium and Acinetobacter were most abundant among the Gram-negative taxa. At the second time point, samples of the same farms were analyzed via both cultivation (1,500 individual colonies each) and high-throughput 16S rRNA gene amplicon sequencing. The latter revealed a threefold higher biodiversity at the genus level, as anaerobic or fastidious species were also detected. However, cultivation identified genera not captured by sequencing, indicating that both approaches are complementary. Using amplicon sequencing, the relative abundance of a few genera was distorted, which seems to be an artifact of sample preparation. Therefore, attention needs to be paid to the library preparation procedure with special emphasis on cell lysis and PCR.

Fundicoccus ignavus gen. nov., sp. nov., a novel genus of the family Aerococcaceae isolated from bulk tank milk.

Three strains of a Gram-stain-positive, catalase-negative, facultative anaerobic, and coccoid species were isolated from German bulk tank milk. Phylogenetic analyses based on the 16S rRNA gene sequences indicated that the three strains (WS4937T, WS4759 and WS5303) constitute an independent phylogenetic lineage within the family Aerococcaceae with Facklamia hominis CCUG 36813T (93.7-94.1 %) and Eremococcus coleocola M1831/95/2T (93.5 %) as most closely related type species. The unclassified strains demonstrated variable growth with 6.5 % (w/v) NaCl and tolerated pH 6.5-9.5. Growth was observed from 12 to 39 °C. Their cell-wall peptidoglycan belongs to the A1α type (l-Lys-direct) consisting of alanine, glutamic acid and lysine. The predominant fatty acids were C16 : 1 ω9c, C16 : 0 and C18 : 1 ω9c and in the polar lipids profile three glycolipids, a phospholipid, phosphatidylglycerol, phosphoglycolipid and diphosphatidylglycerol were found. The G+C content of strain WS4937T was 37.4 mol% with a genome size of ~3.0 Mb. Based on phylogenetic, phylogenomic and biochemical characterizations, the isolates can be demarcated from all other genera of the family Aerococcaceae and, therefore, the novel genus Fundicoccus gen. nov. is proposed. The type species of the novel genus is Fundicoccus ignavus gen. nov., sp. nov. WS4937T (=DSM 109652T=LMG 31441T).

Pseudomonas haemolytica sp. nov., isolated from raw milk and skimmed milk concentrate.

Two strains, WS 5063T and WS 5067, isolated from raw cow's milk and skimmed milk concentrate, could be affiliated as members of the same, hitherto unknown, Pseudomonas species by 16S rRNA and rpoD gene sequences. Multilocus sequence and average nucleotide identity (ANIm) analyses based on draft genome sequences confirmed the discovery of a novel Pseudomonas species. It was most closely related to Pseudomonas synxantha DSM 18928T with an ANIm of 91.4 %. The DNA G+C content of WS 5063T was 60.0 mol %. Phenotypic characterizations showed that the isolates are rod-shaped, motile, catalase- and oxidase-positive, and aerobic. Growth occurred at 4-34 °C and at pH values of pH 5.5-8.0. Both strains showed strong ß-haemolysis on blood agar. The major cellular polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The dominant quinone was Q-9 (90 %), but noticeable amounts of Q-8 (9 %) and traces of Q-7 were also detected. Fatty acid profiles were typical for Pseudomonas species and exhibited C16 : 0 as a major component. Based on these results, we conclude that both strains belong to a novel species, for which the name Pseudomonas haemolytica sp. nov. is proposed. The type strain is WS 5063T (=DSM 108987T=LMG 31232T) and an additional strain is WS 5067 (=DSM 108988=LMG 31233).

Pseudomonas saxonica sp. nov., isolated from raw milk and skimmed milk concentrate.

A polyphasic approach was used to investigate the taxonomic status of two bacterial strains, WS 5072T and WS 5092, isolated from skimmed milk concentrate and raw cow's milk. The 16S rRNA and rpoD gene sequences affiliated the strains to the same, hitherto unknown, Pseudomonas species. Further examinations of the draft genomes based on multilocus sequence analysis and average nucleotide identity confirmed the presence of a novel Pseudomonas species. It was most closely related to Pseudomonas fragi DSM 3456T with 86.3 % ANIm. The DNA G+C content of strain WS 5072T was 56.3 mol%. Cells were aerobic, Gram-negative, catalase and oxidase positive, rod-shaped and motile. Growth occurred at 4-34 °C, pH 5.5-8.0 and with salt concentrations of up to 7 %. The major cellular polar lipids were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The dominating quinone was Q-9 with 94 %, with noticeable amounts of Q-8 (5 %) and traces of Q-7 and Q-10. Fatty acid profiles showed a composition common for Pseudomonas with the major component C16 : 0. Based on these results, the novel species Pseudomonas saxonica sp. nov. is proposed, with the type strain WS 5072T (=DSM 108989T=LMG 31234T) and the additional strain WS 5092 (=DSM 108990=LMG 31235).

Spoilage of Microfiltered and Pasteurized Extended Shelf Life Milk Is Mainly Induced by Psychrotolerant Spore-Forming Bacteria that often Originate from Recontamination.

Premature spoilage and varying product quality due to microbial contamination still constitute major problems in the production of microfiltered and pasteurized extended shelf life (ESL) milk. Spoilage-associated bacteria may enter the product either as part of the raw milk microbiota or as recontaminants in the dairy plant. To identify spoilage-inducing bacteria and their routes of entry, we analyzed end products for their predominant microbiota as well as the prevalence and biodiversity of psychrotolerant spores in bulk tank milk. Process analyses were performed to determine the removal of psychrotolerant spores at each production step. To detect transmission and recontamination events, strain typing was conducted with isolates obtained from all process stages. Microbial counts in 287 ESL milk packages at the end of shelf life were highly diverse ranging from <1 to 7.9 log cfu/mL. In total, 15% of samples were spoiled. High G+C Gram-positive bacteria were the most abundant taxonomic group, but were responsible for only 31% of spoilage. In contrast, psychrotolerant spores were isolated from 55% of spoiled packages. In 90% of samples with pure cultures of Bacillus cereus sensu lato and Paenibacillus spp., counts exceeded 6 log cfu/mL. In bulk tank milk, the concentration of psychrotolerant spores was low, accounting for merely 0.5 ± 0.8 MPN/mL. Paenibacillus amylolyticus/xylanexedens was by far the most dominant species in bulk tank milk (48% of all isolates), but was never detected in ESL milk, pointing to efficient removal during manufacturing. Six large-scale process analyses confirmed a high removal rate for psychrotolerant spores (reduction by nearly 4 log-units). B. cereus sensu lato, on the contrary, was frequently found in spoiled end products, but was rarely detected in bulk tank milk. Due to low counts in bulk tank samples and efficient spore removal during production, we suggest that shelf life is influenced only to a minor extent by raw-milk-associated factors. In contrast, recontamination with spores, particularly from the B. cereus complex, seems to occur. To enhance milk quality throughout the entire shelf life, improved plant sanitation and disinfection that target the elimination of spores are necessary.