Non-canonical transcriptional start sites in E. coli O157:H7 EDL933 are regulated and appear in surprisingly high numbers.

Analysis of genome wide transcription start sites (TSSs) revealed an unexpected complexity since not only canonical TSS of annotated genes are recognized by RNA polymerase. Non-canonical TSS were detected antisense to, or within, annotated genes as well new intergenic (orphan) TSS, not associated with known genes. Previously, it was hypothesized that many such signals represent noise or pervasive transcription, not associated with a biological function. Here, a modified Cappable-seq protocol allows determining the primary transcriptome of the enterohemorrhagic E. coli O157:H7 EDL933 (EHEC). We used four different growth media, both in exponential and stationary growth phase, replicated each thrice. This yielded 19,975 EHEC canonical and non-canonical TSS, which reproducibly occurring in three biological replicates. This questions the hypothesis of experimental noise or pervasive transcription. Accordingly, conserved promoter motifs were found upstream indicating proper TSSs. More than 50% of 5,567 canonical and between 32% and 47% of 10,355 non-canonical TSS were differentially expressed in different media and growth phases, providing evidence for a potential biological function also of non-canonical TSS. Thus, reproducible and environmentally regulated expression suggests that a substantial number of the non-canonical TSSs may be of unknown function rather than being the result of noise or pervasive transcription.

Unified Workflow for the Rapid and In-Depth Characterization of Bacterial Proteomes.

Bacteria are the most abundant and diverse organisms among the kingdoms of life. Due to this excessive variance, finding a unified, comprehensive, and safe workflow for quantitative bacterial proteomics is challenging. In this study, we have systematically evaluated and optimized sample preparation, mass spectrometric data acquisition, and data analysis strategies in bacterial proteomics. We investigated workflow performances on six representative species with highly different physiologic properties to mimic bacterial diversity. The best sample preparation strategy was a cell lysis protocol in 100% trifluoroacetic acid followed by an in-solution digest. Peptides were separated on a 30-min linear microflow liquid chromatography gradient and analyzed in data-independent acquisition mode. Data analysis was performed with DIA-NN using a predicted spectral library. Performance was evaluated according to the number of identified proteins, quantitative precision, throughput, costs, and biological safety. With this rapid workflow, over 40% of all encoded genes were detected per bacterial species. We demonstrated the general applicability of our workflow on a set of 23 taxonomically and physiologically diverse bacterial species. We could confidently identify over 45,000 proteins in the combined dataset, of which 30,000 have not been experimentally validated before. Our work thereby provides a valuable resource for the microbial scientific community. Finally, we grew Escherichia coli and Bacillus cereus in replicates under 12 different cultivation conditions to demonstrate the high-throughput suitability of the workflow. The proteomic workflow we present in this manuscript does not require any specialized equipment or commercial software and can be easily applied by other laboratories to support and accelerate the proteomic exploration of the bacterial kingdom.

Three Novel Antisense Overlapping Genes in E. coli O157:H7 EDL933.

The abundance of long overlapping genes in prokaryotic genomes is likely to be significantly underestimated. To date, only a few examples of such genes are fully established. Using RNA sequencing and ribosome profiling, we found expression of novel overlapping open reading frames in Escherichia coli O157:H7 EDL933 (EHEC). Indeed, the overlapping candidate genes are equipped with typical structural elements required for transcription and translation, i.e., promoters, transcription start sites, as well as terminators, all of which were experimentally verified. Translationally arrested mutants, unable to produce the overlapping encoded protein, were found to have a growth disadvantage when grown competitively against the wild type. Thus, the phenotypes found imply biological functionality of the genes at the level of proteins produced. The addition of 3 more examples of prokaryotic overlapping genes to the currently limited, yet constantly growing pool of such genes emphasizes the underestimated coding capacity of bacterial genomes. IMPORTANCE The abundance of long overlapping genes in prokaryotic genomes is likely to be significantly underestimated, since such genes are not allowed in genome annotations. However, ribosome profiling catches mRNA in the moment of being template for protein production. Using this technique and subsequent experiments, we verified 3 novel overlapping genes encoded in antisense of known genes. This adds more examples of prokaryotic overlapping genes to the currently limited, yet constantly growing pool of such genes.

Spotlight on alternative frame coding: Two long overlapping genes in Pseudomonas aeruginosa are translated and under purifying selection.

The existence of overlapping genes (OLGs) with significant coding overlaps revolutionizes our understanding of genomic complexity. We report two exceptionally long (957 nt and 1536 nt), evolutionarily novel, translated antisense open reading frames (ORFs) embedded within annotated genes in the pathogenic Gram-negative bacterium Pseudomonas aeruginosa. Both OLG pairs show sequence features consistent with being genes and transcriptional signals in RNA sequencing. Translation of both OLGs was confirmed by ribosome profiling and mass spectrometry. Quantitative proteomics of samples taken during different phases of growth revealed regulation of protein abundances, implying biological functionality. Both OLGs are taxonomically restricted, and likely arose by overprinting within the genus. Evidence for purifying selection further supports functionality. The OLGs reported here, designated olg1 and olg2, are the longest yet proposed in prokaryotes and are among the best attested in terms of translation and evolutionary constraint. These results highlight a potentially large unexplored dimension of prokaryotic genomes.

Biological factors in the synthetic construction of overlapping genes.

BACKGROUND: Overlapping genes (OLGs) with long protein-coding overlapping sequences are disallowed by standard genome annotation programs, outside of viruses. Recently however they have been discovered in Archaea, diverse Bacteria, and Mammals. The biological factors underlying life's ability to create overlapping genes require more study, and may have important applications in understanding evolution and in biotechnology. A previous study claimed that protein domains from viruses were much better suited to forming overlaps than those from other cellular organisms - in this study we assessed this claim, in order to discover what might underlie taxonomic differences in the creation of gene overlaps. RESULTS: After overlapping arbitrary Pfam domain pairs and evaluating them with Hidden Markov Models we find OLG construction to be much less constrained than expected. For instance, close to 10% of the constructed sequences cannot be distinguished from typical sequences in their protein family. Most are also indistinguishable from natural protein sequences regarding identity and secondary structure. Surprisingly, contrary to a previous study, virus domains were much less suitable for designing OLGs than bacterial or eukaryotic domains were. In general, the amount of amino acid change required to force a domain to overlap is approximately equal to the variation observed within a typical domain family. The resulting high similarity between natural sequences and those altered so as to overlap is mostly due to the combination of high redundancy in the genetic code and the evolutionary exchangeability of many amino acids. CONCLUSIONS: Synthetic overlapping genes which closely resemble natural gene sequences, as measured by HMM profiles, are remarkably easy to construct, and most arbitrary domain pairs can be altered so as to overlap while retaining high similarity to the original sequences. Future work however will need to assess important factors not considered such as intragenic interactions which affect protein folding. While the analysis here is not sufficient to guarantee functional folding proteins, further analysis of constructed OLGs will improve our understanding of the origin of these remarkable genetic elements across life and opens up exciting possibilities for synthetic biology.

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.

A Strong Synergy Between the Thiopeptide Bacteriocin Micrococcin P1 and Rifampicin Against MRSA in a Murine Skin Infection Model.

Antibiotic-resistant bacterial pathogens have become a serious threat worldwide. One of these pathogens is methicillin-resistant Staphylococcus aureus (MRSA), a major cause of skin and soft tissue infections. In this study we identified a strain of Staphylococcus equorum producing a substance with high antimicrobial activity against many Gram-positive bacteria, including MRSA. By mass spectrometry and whole genome sequencing the antimicrobial substance was identified as the thiopeptide bacteriocin micrococcin P1 (MP1). Based on its properties we developed a one-step purification protocol resulting in high yield (15 mg/L) and high purity (98%) of MP1. For shorter incubation times (5-7 h) MP1 was very potent against MRSA but the inhibitory effect was overshadowed by resistance development during longer incubation time (24h or more). To overcome this problem a synergy study was performed with a number of commercially available antibiotics. Among the antibiotics tested, the combination of MP1 and rifampicin gave the best synergistic effect, with MIC values 25 and 60 times lower than for the individual drugs, respectively. To assess the therapeutic potential of the MP1-rifampicin combination, we used a murine skin infection model based on the use of the multidrug-resistant luciferase-tagged MRSA strain Xen31. As expected, neither of the single antimicrobials (MP1 or rifampicin) could eradicate Xen31 from the wounds. By contrary, the MP1-rifampicin combination was efficient not only to eradicate but also to prevent the recurrence of Xen31 infection. Furthermore, compared to fucidin cream, which is commonly used in skin infection treatments, MP1-rifampicin combination was superior in terms of preventing resistance development. Our results show that combining MP1, and probably other thiopeptides, with antibiotics can be a promising strategy to treat SSTIs caused by MRSA and likely many other Gram-positive bacteria.

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.

Simultaneous quantification of the most common and proteolytic Pseudomonas species in raw milk by multiplex qPCR.

The heat-stable peptidase AprX, secreted by psychrotolerant Pseudomonas species in raw milk, is a major cause of destabilization and premature spoilage of ultra-high temperature (UHT) milk and milk products. To enable rapid detection and quantification of seven frequent and proteolytic Pseudomonas species (P. proteolytica, P. gessardii, P. lactis, P. fluorescens, P. protegens, P. lundensis, and P. fragi) in raw milk, we developed two triplex qPCR assays taking into account species-dependent differences in AprX activity. Besides five species-specific hydrolysis probes, targeting the aprX gene, a universal rpoB probe was included in the assay to determine the total Pseudomonas counts. For all six probes, linear regression lines between Cq value and target DNA concentration were obtained in singleplex as well as in multiplex approaches, yielding R2 values of > 0.975 and amplification efficiencies of 85-97%. Moreover, high specificity was determined using genomic DNA of 75 Pseudomonas strains, assigned to 57 species, and 40 other bacterial species as templates in the qPCR. Quantification of the target species and total Pseudomonas counts resulted in linear detection ranges of approx. 103-107 cfu/ml, which correspond well to common Pseudomonas counts in raw milk. Application of the assay using 60 raw milk samples from different dairies showed good agreement of total Pseudomonas counts calculated by qPCR with cell counts derived from cultivation. Furthermore, a remarkably high variability regarding the species composition was observed for each milk sample, whereby P. lundensis and P. proteolytica/P. gessardii were the predominant species detected. KEY POINTS: • Multiplex qPCR for quantification of seven proteolytic Pseudomonas species and total Pseudomonas counts in raw milk • High specificity and sensitivity via hydrolysis probes against aprX and rpoB • Rapid method to determine Pseudomonas contamination in raw milk and predict spoilage potential.

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.

High counts of thermophilic spore formers in dairy powders originate from persisting strains in processing lines.

During the last decades, thermophilic spore counts became a very important quality parameter for manufacturers with regard to powdered dairy products. Low-spore count powders are highly demanded but challenging to produce when high production volume and long process times are intended. In this study a detailed monitoring of microbial levels in three skim milk powder plants was conducted. Anoxybacillus flavithermus was found to be primarily responsible for increased spore levels with increasing spore numbers being detected after 6-8 h already during initial processing steps. Simultaneously, the species composition shifted from a diverse bulk tank milk microbiota where different Bacillus species represented around 90% of the thermophilic bacteria to a dominance of A. flavithermus in the end product. The analysis of A. flavithermus isolates from different powder batches with RAPD PCR revealed recurring patterns in each of the eight German manufacturers sampled over several months. The high relatedness of isolates exhibiting identical RAPD patterns was exemplified by cgMLST based on whole genome sequences. We assume that A. flavithermus strains persisted in production plants and were not eliminated by cleaning. It is concluded that such persisting strains recurrently recontaminated subsequent powder productions. The data highlight that a targeted optimization of cleaning and disinfection procedures is the most promising measure to effectively reduce thermophilic spore counts in German dairy powders.

Are Antisense Proteins in Prokaryotes Functional?

Many prokaryotic RNAs are transcribed from loci outside of annotated protein coding genes. Across bacterial species hundreds of short open reading frames antisense to annotated genes show evidence of both transcription and translation, for instance in ribosome profiling data. Determining the functional fraction of these protein products awaits further research, including insights from studies of molecular interactions and detailed evolutionary analysis. There are multiple lines of evidence, however, that many of these newly discovered proteins are of use to the organism. Condition-specific phenotypes have been characterized for a few. These proteins should be added to genome annotations, and the methods for predicting them standardized. Evolutionary analysis of these typically young sequences also may provide important insights into gene evolution. This research should be prioritized for its exciting potential to uncover large numbers of novel proteins with extremely diverse potential practical uses, including applications in synthetic biology and responding to pathogens.

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).

Genetic Organization of the aprX-lipA2 Operon Affects the Proteolytic Potential of Pseudomonas Species in Milk.

Psychrotolerant Pseudomonas species are a main cause of proteolytic spoilage of ultra-high temperature (UHT) milk products due to the secretion of the heat-resistant metallopeptidase AprX, which is encoded by the first gene of the aprX-lipA2 operon. While the proteolytic property has been characterized for many different Pseudomonas isolates, the underlying aprX-lipA2 gene organization was only described for a few strains so far. In this study, the phylogenomic analysis of 185 Pseudomonas type strains revealed that the presence of aprX is strongly associated to a monophylum composed of 81 species, of which 83% carried the aprX locus. Furthermore, almost all type strains of known milk-relevant species were shown to be members of the three monophyletic groups P. fluorescens, P. gessardii, and P. fragi. In total, 22 different types of aprX-lipA2 genetic organizations were identified in the genus, whereby 31% of the species tested carried the type 1 operon structure consisting of eight genes (aprXIDEF prtAB lipA2). Other genetic structures differed from type 1 mainly in the presence and location of genes coding for two lipases (lipA1 and lipA2) and putative autotransporters (prtA and prtB). The peptidase activity of 129 strains, as determined on skim milk agar and in UHT-milk, correlated largely with different aprX-lipA2 gene compositions. Particularly, isolates harboring the type 1 operon were highly proteolytic, while strains with other operon types, especially ones lacking prtA and prtB, exhibited significantly lower peptidase activities. In conclusion, the phylogenomic position and the aprX-lipA2 gene organization specify the proteolytic potential of Pseudomonas isolates. In addition, however, an interplay of several environmental factors and intrinsic traits influences production and activity of AprX, leading to strain-specific proteolytic phenotypes.

A Novel pH-Regulated, Unusual 603 bp Overlapping Protein Coding Gene pop Is Encoded Antisense to ompA in Escherichia coli O157:H7 (EHEC).

Antisense transcription is well known in bacteria. However, translation of antisense RNAs is typically not considered, as the implied overlapping coding at a DNA locus is assumed to be highly improbable. Therefore, such overlapping genes are systematically excluded in prokaryotic genome annotation. Here we report an exceptional 603 bp long open reading frame completely embedded in antisense to the gene of the outer membrane protein ompA. An active σ70 promoter, transcription start site (TSS), Shine-Dalgarno motif and rho-independent terminator were experimentally validated, providing evidence that this open reading frame has all the structural features of a functional gene. Furthermore, ribosomal profiling revealed translation of the mRNA, the protein was detected in Western blots and a pH-dependent phenotype conferred by the protein was shown in competitive overexpression growth experiments of a translationally arrested mutant versus wild type. We designate this novel gene pop (pH-regulated overlapping protein-coding gene), thus adding another example to the growing list of overlapping, protein coding genes in bacteria.

Brevilactibacter flavus gen. nov., sp. nov., a novel bacterium of the family Propionibacteriaceae isolated from raw milk and dairy products and reclassification of Propioniciclava sinopodophylli as Brevilactibacter sinopodophylli comb. nov.

Eight facultatively anaerobic rod-shaped bacteria were isolated from raw milk and two other dairy products. Results of phylogenetic analyses based on 16S rRNA gene sequences showed that the isolates are placed in a distinct lineage within the family Propionibacteriaceae with Propioniciclava sinopodophylli and Propioniciclava tarda as the closest relatives (94.6 and 93.5 % similarity, respectively). The cell-wall peptidoglycan contained meso-diaminopimelic acid, alanine and glutamic acid and was of the A1γ type (meso-DAP-direct). The major cellular fatty acid was anteiso-C15 : 0 and the major polar lipids were diphosphatidylglycerol, phosphatidyglycerol and three unidentified glycolipids. The quinone system contained predominantly menaquinone MK-9(H4). The G+C content of the genomic DNA of strain VG341T was 67.7 mol%. The whole-cell sugar pattern contained ribose, rhamnose, arabinose and galactose. On the basis of phenotypic and genetic data, eight strains (VG341T, WS4684, WS4769, WS 4882, WS4883, WS4901, WS4902 and WS4904) are proposed to be classified as members of a novel species in a new genus of the family Propionibacteriaceae, for which the name Brevilactibacter flavus gen. nov., sp. nov. is proposed. The type strain is VG341T (=WS4900T=DSM 100885T=LMG 29089T) and seven additional strains are WS4684, WS4769, WS4882, WS4883, WS4901, WS4902 and WS4904. Furthermore, we propose the reclassification of P. sinopodophylli as Brevilactibacter sinopodophylli comb. nov.

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).

Resistance of thermophilic spore formers isolated from milk and whey products towards cleaning-in-place conditions: Influence of pH, temperature and milk residues.

The occurrence of thermophilic spore formers in dairy powders is a major concern for producers worldwide. This study aims to investigate the resistance of thermophilic endospores towards cleaning solutions typically used for cleaning-in-place in dairy manufacturing plants. From eleven tested strains, all were able to survive an alkaline treatment (NaOH) at 65 °C for 10 min (0.5%), whereas at concentrations of 2% eight strains withstood the treatment. Acid solutions were more sporicidal. At 0.5% of HNO3, only three strains survived the treatment. Milk impurities reduced the inactivation effect of the NaOH solutions towards thermophilic spore formers. For two selected strains, a detailed kinetic inactivation in NaOH and HNO3 solutions at different temperatures was performed and non-log-linear inactivation curves were observed. This study highlights the risk of reusing cleaning solutions in dairies.