Phenotypic and genomic characterization of Klebsiella pneumoniae subsp. pneumoniae and Rahnella inusitata strains reveals no clear association between genetic content and ropy phenotype
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Ropy defect in fluid milk
Ropy defect of pasteurized fluid milk is a type of spoilage which manifests itself by an increased viscosity, slimy body, and string-like flow during pouring. This defect has, among other causes, been attributed to the growth, proliferation and exopolysaccharide production by coliform bacteria, which are most commonly introduced in milk as post-pasteurization contaminants. As we identified both Klebsiella pneumoniae subsp. pneumoniae and Rahnella inusitata that were linked to a ropy defect, the goal of this study was to characterize three K. pneumoniae subsp. pneumoniae strains and two R. inusitata for (i) their ability to grow and cause ropy defect in milk at 6 and 21°C and to (ii) probe the genetic basis for observed ropy phenotype. While all K. pneumoniae subsp. pneumoniae and R. inusitata strains showed net growth of >4 log10 over 48 h in UHT milk at 21°C, only R. inusitata strains displayed growth during 28-day incubation period at 6°C (>6 log10). Two out of three K. pneumoniae subsp. pneumoniae strains were capable of causing the ropy defect in milk at 21°C, as supported by an increase in the viscosity of milk and string-like flow during pouring; these two strains were originally isolated from raw milk. Only one R. inusitata strains was able to cause the ropy defect in milk; this strain was able to cause the defect at both 6 and 21°C, and was originally isolated from a pasteurized milk. These findings suggest that the potential of K. pneumoniae subsp. pneumoniae and R. inusitata to cause ropy defect in milk is a strain-dependent characteristic. Comparative genomics provided no definitive answer on genetic basis for the ropy phenotype. However, for K. pneumoniae subsp. pneumoniae, genes rffG, rffH, rfbD, and rfbC involved in biosynthesis and secretion of enterobacterial common antigen (ECA) could only be found in the two strains that produced ropy defect, and for R. inusitata a set of two glycosyltransferase and flippase genes involved in nucleotide sugar biosynthesis and export could only be identified in the ropy strain. While these results provide some initial information for potential markers for strains that can cause ropy milk, the relationship between genetic content and ropiness in milk remains poorly understood and merits further investigation.
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Supplementary Materials: (i) Supplementary Figures: - Supplementary Figure 1: Venn diagram of core, accessory and unique gene families identified in Rahnella inusitata FSL A6-0213 and FSL J3-0059 genomes. The number of core genes shared by all strains is in the center (4,141). The number in non-overlapping portions of each oval represents the size of unique genes. RP+ represents positive ropy phenotype. RP- represents negative ropy phenotype. (ii) Supplementary Tables: - Supplementary Table 1: General and specific genome features of three Klebsiella pneumoniae subsp. pneumoniae and two Rahnella inusitata strains. - Supplementary Table 2: Flow rate of milk through 10 mL serological pipette measured during individual growth of three Klebsiella pneumoniae subsp. pneumoniae and two Rahnella inusitata strains. - Supplementary Table 3: Matrix representation of the Average Nucleotide Identity by BLAST (ANIb) in percent between the Klebsiella sp. strains. - Supplementary Table 4: Matrix representation of the Average Nucleotide Identity by BLAST (ANIb) in percent between the Rahnella inusitata strains. - Supplementary Table 5: Gene presence-absence in (A) Klebsiella pneumoniae subsp. pneumoniae pangenome and (B) Rahnella inusitata pangenome. Core genes are shown in red, and accessory genes are shown in blue.
This project was supported by the New York State Milk Promotion Advisory Board through the New York State Department of Agriculture and Markets (Albany, NY). The authors thank the staff and students of the Milk Quality Improvement Program (Cornell University, Ithaca, NY) and the Food Safety Lab (Cornell University, Ithaca, NY) for assistance with this study; specifically, Molly Higgins, Hongyu Ou, and Jasna Kovac. The authors also thank Carmen 651 Moraru (Department of Food Science, Cornell University, Ithaca, NY) for enabling them to measure viscosity of milk samples. The authors acknowledge that N. H. Martin is a section editor for the Journal of Dairy Science.
Journal of Dairy Science
dairy spoilage, ropy milk, Klebsiella, Rahnella, genomics
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