Multiplex-PCR Detection of an Atypical Leuconostoc mesenteroides subsp. jonggajibkimchii Phenotype Dominating the Terminal Spoilage Microbial Association of a Fresh Greek Whey Cheese Stored at 4 °C in Vacuum
Abstract
:1. Introduction
2. Materials and Methods
2.1. Whey Cheese Spoilage Isolates and Culture Conditions
2.2. Biochemical Differentiation of the Leuconostoc spp. Isolates
2.3. Differentiation and Identification of the Leuconostoc spp. Isolates by Multiplex-PCR
3. Results
3.1. Biotyping of the Whey Cheese Spoilage Leuconostoc spp. Isolates
3.2. Classification of the Whey Cheese Spoilage Ln. mesenteroides Isolates by Multiplex-PCR—Prevalence of Isolates with a Gene Profile Specific to Ln. mesenteroides subsp. jonggajibkimchii
Species Identification/ Isolate Code | Strain Biotype | LAra 4 | Rib 5 | DXyl 6 | Gal 10 | Glu 11 | Fru 12 | Mne 13 | Man 18 | MDG 21 | NAG 22 | Arb 24 | Esc 25 | Sal 26 | Mal 28 | Lac 29 | Mel 30 | Sac 31 | Tre 32 | Raf 35 | Tur 40 |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Ln. mesenteroides | |||||||||||||||||||||
WM105 | L2C | + | + | + | + | + | + | + | (+)d | + | + | (+) | + | + | + | + | + | + | + | + | + |
WM121 | L3 | + | + | + | + | + | + | + | (+)d | + | + | (+) | + | + | + | + | - | + | + | - | + |
WM119 | L1E | - | - | + | + | + | + | + | (+)d | + | + | (+) | (+) | + | + | + | - | + | + | - | + |
WM106 | L1F | - | - | + | + | + | + | + | - | + | + | - | (+) | + | + | + | + | + | + | - | + |
WM107 | L4B | - | - | + | + | + | + | + | - | + | + | - | (+) | - | + | + | - | + | + | - | + |
WM117 | L1D | - | - | + | + | + | + | + | - | + | + | - | (+) | - | + | + | - | + | + | - | + |
WM137 | L1C | - | - | + | + | + | + | + | - | - | + | - | - | - | + | + | - | + | + | - | - |
WM136 | L1B | - | - | + | + | + | + | + | - | - | + | - | - | - | - | + | - | + | + | - | - |
WM138 | L1B | - | - | + | + | + | + | + | - | - | + | - | - | - | - | + | - | + | + | - | - |
WM147 | L1B | - | - | + | + | + | + | + | - | - | + | - | - | - | - | + | - | + | + | - | - |
WM151 | L1B | - | - | + | + | + | + | + | - | - | + | - | - | - | - | + | - | + | + | - | - |
WM153 | L1B | - | - | + | + | + | + | + | - | - | + | - | - | - | - | + | - | + | + | - | - |
WM108 | L4A | - | - | - | + | + | + | + | - | - | + | - | - | - | - | + | - | + | + | - | - |
WM124 | L1A | - | - | - | + | + | + | + | - | - | + | - | - | - | - | + | - | + | + | - | - |
Ln. lactis | |||||||||||||||||||||
WM118 | L5 | - | - | - | + | + | + | + | - | - | + | - | - | - | + | + | + | + | - | + | - |
WM129 | L5 | - | - | - | + | + | + | + | - | - | + | - | - | - | + | + | + | + | - | + | - |
Species Identification | Strain Code | Target Genes Detected by Multiplex-PCR | Subspecies Identification | Reference | Multiplex Profile | Basic Biotype | Updated Biotype | |||
---|---|---|---|---|---|---|---|---|---|---|
Reference strains (literature/our previous data) | rpoB | araA | dsr | sorA | ||||||
Ln. mesenteroides | ATCC 19254T | + | - | - | - | cremoris | [46] | S1 | NA | NA |
Ln. mesenteroides | DSM 20484T | + | - | + | - | dextranicum | [46] | S2 | NA | NA |
Ln. mesenteroides | DRC1506T | + | + | + | - | jonggajibkimchii | [46] | S3 | NA | NA |
Ln. mesenteroides | ATCC 8293T | + | + | + | + | mesenteroides | [46] | S4 | NA | NA |
Ln. mesenteroides | ACA-DC 0750 | + | + | + | + | mesenteroides | [45] | S4 | NA | NA |
Ln. mesenteroides | ACA-DC 0493 | + | - | + | - | dextranicum | [45] | S2 | NA | NA |
Ln. mesenteroides | ACA-DC 0231 | + | - | + | + | dextranicum (atypical) | [45] | S5 | NA | NA |
Anthotyros cheese strains | This study | |||||||||
Ln. mesenteroides | WM124 | + | - | + | + | dextranicum (atypical) | S5 | L1 | L1A | |
Ln. mesenteroides | WM109A | + | + | + | - | jonggajibkimchii | S3 | L1 | L1B | |
Ln. mesenteroides | WM136 | + | + | + | - | jonggajibkimchii | S3 | L1 | L1B | |
Ln. mesenteroides | WM138 | + | + | + | - | jonggajibkimchii | S3 | L1 | L1B | |
Ln. mesenteroides | WM147 | + | + | + | - | jonggajibkimchii | S3 | L1 | L1B | |
Ln. mesenteroides | WM151 | + | + | + | - | jonggajibkimchii | S3 | L1 | L1B | |
Ln. mesenteroides | WM153 | + | + | + | - | jonggajibkimchii | S3 | L1 | L1B | |
Ln. mesenteroides | WM137 | + | + | + | - | jonggajibkimchii | S3 | L1 | L1C | |
Ln. mesenteroides | WM109B | + | - | + | + | dextranicum (atypical) | S5 | L1 | L1D | |
Ln. mesenteroides | WM117 | + | - | + | + | dextranicum (atypical) | S5 | L1 | L1D | |
Ln. mesenteroides | WM119 | + | - | + | + | dextranicum (atypical) | S5 | L1 | L1E | |
Ln. mesenteroides | WM123 | + | - | + | + | dextranicum (atypical) | S5 | L1 | L1E | |
Ln. mesenteroides | WM125A | + | - | + | + | dextranicum (atypical) | S5 | L1 | L1E | |
Ln. mesenteroides | WM125B | + | - | + | + | dextranicum (atypical) | S5 | L1 | L1E | |
Ln. mesenteroides | WM106 | + | - | + | + | dextranicum (atypical) | S5 | L1 | L1F | |
Ln. mesenteroides | WM122A | + | + | + | + | mesenteroides | S4 | L2 | L2A | |
Ln. mesenteroides | WM110A | + | + | + | - | jonggajibkimchii | S3 | L2 | L2B | |
Ln. mesenteroides | WM110B | + | + | + | - | jonggajibkimchii | S3 | L2 | L2B | |
Ln. mesenteroides | WM103 | + | + | + | + | mesenteroides | S4 | L2 | L2C | |
Ln. mesenteroides | WM105 | + | + | + | + | mesenteroides | S4 | L2 | L2C | |
Ln. mesenteroides | WM122B | + | + | + | + | mesenteroides | S4 | L2 | L2C | |
Ln. mesenteroides | WM121 | + | - | + | + | dextranicum (atypical) | S5 | L3 | L3 | |
Ln. mesenteroides | WM108 | + | + | + | - | jonggajibkimchii | S3 | L4 | L4A | |
Ln. mesenteroides | WM107 | + | - | + | + | dextranicum (atypical) | S5 | L4 | L4B | |
Ln. lactis | WM118 | - | - | - | - | None (N/A) | No bands | L5 | L5 | |
Ln. lactis | WM129 | - | - | - | - | None (N/A) | No bands | L5 | L5 |
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Group/ Isolate Biotype | Cheese Batch/ Treatment | Isolate Code 2 | Species Identification | Closest Ref. Strain in BLAST | 16S rRNA Gene Seq Similarity |
---|---|---|---|---|---|
L1 | C/CN | WM106 | Ln. mesenteroides | MT545072.1 | 100 |
C/CN | WM109A | Ln. mesenteroides | NT | - | |
C/CN | WM109B | Ln. mesenteroides | MT545072.1 | 100 | |
C/Ent+ | WM117 | Ln. mesenteroides | NT | - | |
C/Ent+ | WM119 | Ln. mesenteroides | NT | - | |
C/Ent+ | WM123 | Ln. mesenteroides | MT545101.1 | 100 | |
C/Ent+ | WM124 | Ln. mesenteroides | NT | - | |
C/Ent+ | WM125A | Ln. mesenteroides | NT | - | |
C/Ent+ | WM125B | Ln. mesenteroides | NT | - | |
D/CN | WM136 | Ln. mesenteroides | MT545072.1 | 100 | |
D/CN | WM137 | Ln. mesenteroides | MT545072.1 | 100 | |
D/CN | WM138 | Ln. mesenteroides | NT | - | |
D/Ent+ | WM147 | Ln. mesenteroides | NT | - | |
D/Ent+ | WM151 | Ln. mesenteroides | NT | - | |
D/Ent+ | WM153 | Ln. mesenteroides | MT545072.1 | 100 | |
L2 | C/CN | WM103 | Ln. mesenteroides | NT | - |
C/CN | WM105 | Ln. mesenteroides | MT545072.1 | 100 | |
C/CN | WM110A | Ln. mesenteroides | MT545113.1 | 100 | |
C/CN | WM110B | Ln. mesenteroides | NT | - | |
C/Ent+ | WM122A | Ln. mesenteroides | MT545072.1 | 100 | |
C/Ent+ | WM122B | Ln. mesenteroides | NT | - | |
L3 | C/Ent+ | WM121 | Ln. mesenteroides | MT545113.1 | 100 |
L4 | C/CN | WM107 | Ln. mesenteroides | MT545113.1 | 100 |
C/CN | WM108 | Ln. mesenteroides | MT545072.1 | 100 | |
L5 | C/Ent+ | WM118 | Ln. lactis | MF354765.1 | 100 |
C/Ent+ | WM129 | Ln. lactis | NT | - |
Gene | Primer | Sequence (5′-3′) | Amplicon Size (bp) | Annealing Temperature (°C) | Reference |
---|---|---|---|---|---|
rpoB | rpob-F | GTCCGCATTGATCGCACGC | 952 | 60 | Ricciardi et al. [46] |
rpob-R | CACCCGGTCCAAGAGCTGAC | ||||
araA | L-ara-F | TTTGGCTGGACGGTTGACT | 744 | ||
L-ara-R | TGTTGTGTGATGTCCGCCAC | ||||
dsr | dextran-F | TGGCACCATTACCATAACGAACT | 549 | ||
dextran-R | TGCCAGCAGTCGATCAATATGG | ||||
sorA | PTS-sorb-F | GTGCCTTACTCCCCTGTGTAG | 253 | ||
PTS-sorb-R | TCCTCGTCTTCCTCATCATCGT |
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Sameli, N.; Sioziou, E.; Bosnea, L.; Paramithiotis, S.; Samelis, J. Multiplex-PCR Detection of an Atypical Leuconostoc mesenteroides subsp. jonggajibkimchii Phenotype Dominating the Terminal Spoilage Microbial Association of a Fresh Greek Whey Cheese Stored at 4 °C in Vacuum. Appl. Microbiol. 2024, 4, 1124-1141. https://doi.org/10.3390/applmicrobiol4030076
Sameli N, Sioziou E, Bosnea L, Paramithiotis S, Samelis J. Multiplex-PCR Detection of an Atypical Leuconostoc mesenteroides subsp. jonggajibkimchii Phenotype Dominating the Terminal Spoilage Microbial Association of a Fresh Greek Whey Cheese Stored at 4 °C in Vacuum. Applied Microbiology. 2024; 4(3):1124-1141. https://doi.org/10.3390/applmicrobiol4030076
Chicago/Turabian StyleSameli, Nikoletta, Eleni Sioziou, Loulouda Bosnea, Spiros Paramithiotis, and John Samelis. 2024. "Multiplex-PCR Detection of an Atypical Leuconostoc mesenteroides subsp. jonggajibkimchii Phenotype Dominating the Terminal Spoilage Microbial Association of a Fresh Greek Whey Cheese Stored at 4 °C in Vacuum" Applied Microbiology 4, no. 3: 1124-1141. https://doi.org/10.3390/applmicrobiol4030076