sp. nov., a novel proteolytic species isolated from cream Free

Abstract

During a study investigating the microbiota of raw milk and its semi-finished products, strains WS 5106 and WS 5096 were isolated from cream and skimmed milk concentrate. They could be assigned to the genus 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 species within the subgroup. With pairwise ANIm values of 90.1 and 89.8 %, WS 5106 was most closely related to CECT 9765 and CECT 9766. The G+C content of strain WS 5106 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 , with C 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, sp. nov., with WS 5106 (=DSM 111143=LMG 31863) as type strain and WS 5096 (=DSM 111129=LMG 31864) as an additional strain.

Keyword(s): peptidase , Pseudomonas and raw milk
Funding
This study was supported by the:
  • Forschungskreis der Ernährungsindustrie (Award AiF 20027N)
    • Principle Award Recipient: NotApplicable
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2020-12-08
2024-03-28
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References

  1. von Neubeck M, Baur C, Krewinkel M, Stoeckel M, Kranz B et al. Biodiversity of refrigerated raw milk microbiota and their enzymatic spoilage potential. Int J Food Microbiol 2015; 211:57–65 [View Article][PubMed]
    [Google Scholar]
  2. Marchand S, Heylen K, Messens W, Coudijzer K, De Vos P et al. Seasonal influence on heat-resistant proteolytic capacity of Pseudomonas lundensis and Pseudomonas fragi, predominant milk spoilers isolated from Belgian raw milk samples. Environ Microbiol 2009; 11:467–482 [View Article][PubMed]
    [Google Scholar]
  3. de Oliveira GB, Favarin L, Luchese RH, McIntosh D. Psychrotrophic bacteria in milk: how much do we really know?. Braz J Microbiol 2015; 46:313–321 [View Article][PubMed]
    [Google Scholar]
  4. Lafarge V, Ogier J-C, Girard V, Maladen V, Leveau J-Y et al. Raw cow milk bacterial population shifts attributable to refrigeration. Appl Environ Microbiol 2004; 70:5644–5650 [View Article][PubMed]
    [Google Scholar]
  5. Vithanage NR, Dissanayake M, Bolge G, Palombo EA, Yeager TR et al. Biodiversity of culturable psychrotrophic microbiota in raw milk attributable to refrigeration conditions, seasonality and their spoilage potential. Int Dairy J 2016; 57:80–90 [View Article]
    [Google Scholar]
  6. Sørhaug T, Stepaniak L. Psychrotrophs and their enzymes in milk and dairy products: quality aspects. Trends Food Sci Techn 1997; 8:35–41 [View Article]
    [Google Scholar]
  7. Hantsis-Zacharov E, Halpern M. Culturable psychrotrophic bacterial communities in raw milk and their proteolytic and lipolytic traits. Appl Environ Microbiol 2007; 73:7162–7168 [View Article][PubMed]
    [Google Scholar]
  8. Cousin MA. Presence and activity of psychrotrophic microorganisms in milk and dairy products: a review. J Food Prot 1982; 45:172–207 [View Article][PubMed]
    [Google Scholar]
  9. Dogan B, Boor KJ. Genetic diversity and spoilage potentials among Pseudomonas spp. isolated from fluid milk products and dairy processing plants. Appl Environ Microbiol 2003; 69:130–138 [View Article][PubMed]
    [Google Scholar]
  10. Borch E, Kant-Muermans ML, Blixt Y. Bacterial spoilage of meat and cured meat products. Int J Food Microbiol 1996; 33:103–120 [View Article][PubMed]
    [Google Scholar]
  11. Stoeckel M, Lidolt M, Achberger V, Glück C, Krewinkel M et al. Growth of Pseudomonas weihenstephanensis, Pseudomonas proteolytica and Pseudomonas sp. in raw milk: Impact of residual heat-stable enzyme activity on stability of UHT milk during shelf-life. Int Dairy J 2016; 59:20–28 [View Article]
    [Google Scholar]
  12. Qin J, Hu Y, Feng Y, Xaioju L, Zong Z. Pseudomonas sichuanensis sp. nov., isolated from hospital sewage. Int J Syst Evol Microbiol 2019; 69:517–522 [View Article][PubMed]
    [Google Scholar]
  13. Anurat P, Duangmal K, Srisuk N. Pseudomonas mangiferae sp. nov., isolated from bark of mango tree in Thailand. Int J Syst Evol Microbiol 2019; 69:35373543 [View Article][PubMed]
    [Google Scholar]
  14. Hofmann K, Huptas C, Doll EV, Scherer S, Wenning M. Pseudomonas haemolytica sp. nov., isolated from raw milk and skimmed milk concentrate. Int J Syst Evol Microbiol 2020; 70:23392347 [View Article][PubMed]
    [Google Scholar]
  15. Hofmann K, Huptas C, Doll EV, Scherer S, Wenning M. Pseudomonas saxonica sp. nov., isolated from raw milk and skimmed milk concentrate. Int J Syst Evol Microbiol 2020; 70:935–943 [View Article][PubMed]
    [Google Scholar]
  16. Chun J, Oren A, Ventosa A, Christensen H, Arahal DR et al. Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. Int J Syst Evol Microbiol 2018; 68:461–466 [View Article][PubMed]
    [Google Scholar]
  17. Huptas C, Scherer S, Wenning M. Optimized illumina PCR-free library preparation for bacterial whole genome sequencing and analysis of factors influencing de novo assembly. BMC Res Notes 2016; 9:269 [View Article][PubMed]
    [Google Scholar]
  18. Patel RK, Jain M. NGS QC toolkit: a toolkit for quality control of next generation sequencing data. PLoS One 2012; 7:e30619 [View Article][PubMed]
    [Google Scholar]
  19. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 2012; 19:455–477 [View Article][PubMed]
    [Google Scholar]
  20. Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res 2015; 25:1043–1055 [View Article][PubMed]
    [Google Scholar]
  21. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP et al. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res 2016; 44:6614–6624 [View Article][PubMed]
    [Google Scholar]
  22. Haft DH, DiCuccio M, Badretdin A, Brover V, Chetvernin V et al. Refseq: an update on prokaryotic genome annotation and curation. Nucleic Acids Res 2018; 46:D851–D860 [View Article][PubMed]
    [Google Scholar]
  23. Mulet M, Bennasar A, Lalucat J, García-Valdés E. An rpoD-based PCR procedure for the identification of Pseudomonas species and for their detection in environmental samples. Mol Cell Probes 2009; 23:140–147 [View Article][PubMed]
    [Google Scholar]
  24. Chain PSG, Grafham DV, Fulton RS, Fitzgerald MG, Hostetler J et al. Genomics. genome project standards in a new era of sequencing. Science 2009; 326:236–237 [View Article][PubMed]
    [Google Scholar]
  25. Moore ERB, Tindall BJ, Martins Dos Santos VAP, Pieper DH, Ramos JL et al. Nonmedical: Pseudomonas , 3rd ed. New York: Springer; 2006 pp 646–703
    [Google Scholar]
  26. Kim O-S, Cho Y-J, Lee K, Yoon S-H, Kim M et al. Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 2012; 62:716–721 [View Article][PubMed]
    [Google Scholar]
  27. Edgar RC. Muscle: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 2004; 32:1792–1797 [View Article][PubMed]
    [Google Scholar]
  28. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 2014; 30:2068–2069 [View Article][PubMed]
    [Google Scholar]
  29. Bayliss SC, Thorpe HA, Coyle NM, Sheppard SK, Feil EJ. PIRATE: a fast and scalable pangenomics toolbox for clustering diverged orthologues in bacteria. Gigascience 2019; 8: 01 10 2019 [View Article][PubMed]
    [Google Scholar]
  30. Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD et al. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol Biol Evol 2020; 37:1530–1534 [View Article][PubMed]
    [Google Scholar]
  31. Hoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS. UFBoot2: improving the ultrafast bootstrap approximation. Mol Biol Evol 2018; 35:518–522 [View Article][PubMed]
    [Google Scholar]
  32. Letunic I, Bork P. Interactive tree of life (iTOL) V4: recent updates and new developments. Nucleic Acids Res 2019; 47:W256–W259 [View Article][PubMed]
    [Google Scholar]
  33. Richter M, Rosselló-Móra R. Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci USA 2009; 106:19126–19131 [View Article][PubMed]
    [Google Scholar]
  34. Tambong JT, Xu R, Bromfield ESP. Pseudomonas canadensis sp. nov., a biological control agent isolated from a field plot under long-term mineral fertilization. Int J Syst Evol Microbiol 2017; 67:889–895 [View Article][PubMed]
    [Google Scholar]
  35. Iizuka H, Komagata K. New species of Pseudomonas belonged to fluorescent group (studies on the microorganisms of cereal grains. Part V). J Agric Chem Soc Japan 1963; 37:137–141
    [Google Scholar]
  36. Ivanova EP, Gorshkova NM, Sawabe T, Hayashi K, Kalinovskaya NI et al. Pseudomonas extremorientalis sp. nov., isolated from a drinking water reservoir. Int J Syst Evol Microbiol 2002; 52:2113–2120 [View Article][PubMed]
    [Google Scholar]
  37. Vela AI, Gutiérrez MC, Falsen E, Rollán E, Simarro I et al. Pseudomonas simiae sp. nov., isolated from clinical specimens from monkeys (Callithrix geoffroyi). Int J Syst Evol Microbiol 2006; 56:2671–2676 [View Article][PubMed]
    [Google Scholar]
  38. Behrendt U, Ulrich A, Schumann P, Meyer J-M, Spröer C. Pseudomonas lurida sp. nov., a fluorescent species associated with the phyllosphere of grasses. Int J Syst Evol Microbiol 2007; 57:979–985 [View Article][PubMed]
    [Google Scholar]
  39. Munsch P, Alatossava T, Marttinen N, Meyer J-M, Christen R et al. Pseudomonas costantinii sp. nov., another causal agent of brown blotch disease, isolated from cultivated mushroom sporophores in Finland. Int J Syst Evol Microbiol 2002; 52:1973–1983 [View Article][PubMed]
    [Google Scholar]
  40. Skerman VBD, Sneath PHA, McGowan V. Approved Lists of bacterial names. Int J Syst Evol Microbiol 1980; 30:225–420 [View Article]
    [Google Scholar]
  41. Oueslati M, Mulet M, Gomila M, Berge O, Hajlaoui MR et al. New species of pathogenic Pseudomonas isolated from citrus in Tunisia: proposal of Pseudomonas kairouanensis sp. nov. and Pseudomonas nabeulensis sp. nov. Syst Appl Microbiol 2019; 42:348–359 [View Article][PubMed]
    [Google Scholar]
  42. Ryu E. A simple method of differentiation between Gram-positive and Gram-negative organisms without staining. Kitasato Arch Exp Med 1940; 17:58–36
    [Google Scholar]
  43. Xu P, Li W-J, Tang S-K, Zhang Y-Q, Chen G-Z et al. Naxibacter alkalitolerans gen. nov., sp. nov., a novel member of the family 'Oxalobacteraceae' isolated from China. Int J Syst Evol Microbiol 2005; 55:1149–1153 [View Article][PubMed]
    [Google Scholar]
  44. King EO, Ward MK, Raney DE. Two simple media for the demonstration of pyocyanin and fluorescin. J Lab Clin Med 1954; 44:301–307[PubMed]
    [Google Scholar]
  45. Woods RG, Burger M, Beven C-A, Beacham IR. The aprX-lipA operon of Pseudomonas fluorescens B52: a molecular analysis of metalloprotease and lipase production. Microbiology 2001; 147:345–354 [View Article][PubMed]
    [Google Scholar]
  46. Maier C, Huptas C, von Neubeck M, Scherer S, Wenning M et al. Genetic organization of the aprX-lipA2 operon affects the proteolytic potential of Pseudomonas species in milk. Front Microbiol 2020; 11:1190 [View Article][PubMed]
    [Google Scholar]
  47. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959; 37:911–917 [View Article][PubMed]
    [Google Scholar]
  48. Tindall BJ. A comparative study of the lipid composition of Halobacterium saccharovorum from various sources. Syst Appl Microbiol 1990; 13:128–130 [View Article]
    [Google Scholar]
  49. Tindall BJ, Sikorski J, Krieg NR. Phenotypic characterization and the principles of comparative systematics. In Reddy C, Beveridge T, Breznak JA, Marzluf G, Schmidt TM. (editors) Methods for General and Molecular Microbiology Washington, DC: American Society for Microbiology; 2007 pp 330–393
    [Google Scholar]
  50. Tindall BJ. Lipid composition of Halobacterium lacusprofundi . FEMS Microbiol Lett 1990; 66:199–202 [View Article]
    [Google Scholar]
  51. Miller LT. A single derivatization method for bacterial fatty acid methyl esters including hydroxy acids. J Clin Microbiol 1982; 16:584–586
    [Google Scholar]
  52. Kuykendall LD, Roy MA, O'NEILL JJ, Devine TE. Fatty acids, antibiotic resistance, and deoxyribonucleic acid homology groups of Bradyrhizobium japonicum. Int J Syst Bacteriol 1988; 38:358–361 [View Article]
    [Google Scholar]
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