Identification and Characterisation of Spore-Forming Bacteria in Bovine Raw Milk Collected from Four Dairy Farms in New Zealand
Abstract
:1. Introduction
2. Materials and Methods
2.1. Study Sites
2.2. Processing of Raw-Milk Samples
2.3. Bacterial Isolates
2.4. Isolation of Mesophilic Spore-Forming Bacteria from Raw-Milk Samples
2.5. Extraction of DNA
2.6. ERIC PCR Fingerprinting to Identify Unique DNA Patterns in Isolates for Further Analysis
2.7. Amplification and Sequencing of 16S rRNA Gene
2.8. Detection of Toxin Genes Using PCR
2.9. Toxin Production by Clostridium Perfringens
2.10. Screening for Spoilage Potential
3. Results
3.1. Enumeration of Aerobic Spore-Forming Bacteria Present in Raw Milk
3.2. ERIC Profiles and 16S rRNA Sequencing
3.3. Different Aerobic Spore-Forming Bacteria Present in Raw-Milk Samples
3.4. Different Anaerobic Spore-Forming Bacteria Present in Raw Milk
3.5. Detection of Toxin Genes in Bacillus spp. and C. perfringens
3.6. Spoilage Activity
4. Discussion
5. Conclusions
6. Future Directions
Supplementary Materials
Author Contributions
Funding
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primers and Target Genes | Sequence (5′-3′) | Product Size bp | Reference |
---|---|---|---|
LicA-F | GTGCCTGATGTAACGAATG | 735 | [49] |
LicA-R | CACTTCCTGCCATATACC | ||
LicB-F | TGATCAGCCGGCCGTTGTCT | 904 | [49] |
LicB-R | GGCGAATTGTCCGATCATGTCC | ||
LicC-F | GCCTATCTGCCGATTGAC | 1195 | [49] |
LicC-R | TATATGCATCCGGCACCA | ||
cesB-F | GACAAGAGAAATTTCTACGAGCAAGTACAAT | 635 | [50] |
cesB-R | GCAGCCTTCCAATTACTCCTTCTGCCACAGT |
Primers and Target Genes | Sequence (5′-3′) | Product Size bp | Reference |
---|---|---|---|
hblA-F | ATT AAT ACA GGG GAT GGA GAA ACT T | 237 | [51] |
hblA-R | TGA TCC TAA TAC TTC TAG ACG CTT | ||
hblC-F | CCT ATC AAT ACT CTC GCA ACA CCA AT | 386 | [51] |
hblC-R | TTT TCT TGA TTC GTC ATA GCC ATT TCT | ||
hblD-F | AGA TGC TAC AAG ACT TCA AAG GGA AAC TAT | 436 | [51] |
hblD-R | TGA TTA GCA CGA TCT GCT TTC ATA CTT | ||
nheA-F | ATT ACA GGG TTA TTG GTT ACA GCA GT | 475 | [51] |
nheA-R | AAT CTT GCT CCA TAC TCT CTT GGA TGC T | ||
nheB-F | GTG CAG CTG TAG GCG GT | 328 | [51] |
nheB-R | ATG TTT TTC CAG CTA TCT TTC GCA AT | ||
nheC-F | GCG GAT ATT GTA AAG AAT CAA AAT GAG GT | 557 | [51] |
nheC-R | TTT CCA GCT ATC TTT CGC TGT ATG TAA AT | ||
entFM-F | CAA AGA CTT CGT AAC AAA AGG TGG T | 290 | [51] |
entFM-R | TGT TTA CTC CGC CTT TTA CAA ACT T | ||
cytK1-F | ATC GGG CAA AAT GCA AAA ACA CAT | 800 | [51] |
ctyK1-R | ACC CAG TTT GCA GTT CCG AAT GT | ||
cesB-F | GACAAGAGAAATTTCTACGAGCAAGTACAAT | 635 | [50] |
cesB-R | GCAGCCTTCCAATTACTCCTTCTGCCACAGT |
Primers and Target Genes | Sequence (5′-3′) | Product Size bp |
---|---|---|
CPA-F | GTTGATAGCGCAGGACATGTTAAG | 402 |
CPA-R | CATGTAGTCATCTGTTCCAGCATC | |
CPB-F | ACTATACAGACAGATCATTCAACC | 236 |
CPB-R | TTAGGAGCAGTTAGAACTACAGAC | |
CPE-F | ACTGCAACTACTACTCATACTGTG | 541 |
CPE-R | CTGGTGCCTTAATAGAAAGACTCC | |
CPI-F | GCGATGAAAAGCCTACACCACTAC | 317 |
CPI-R | GGTATATCCTCCACGCATATAGTC |
Isolates | LicA | LicB | LicC | cesB |
---|---|---|---|---|
Farm 1, Summer 1 | − | − | + | − |
Farm 1, Summer 5 | + | + | + | − |
Farm 1, Summer 7 | + | − | + | − |
Farm 1, Summer 8 | + | − | + | − |
Farm 1, Summer 9 | − | + | + | − |
Farm 1, Summer 11 | + | − | + | − |
Farm 1, Summer 12 | + | − | − | − |
Farm 1, Summer 13 | + | − | − | − |
Farm 1, Summer 2 (B. pumilus) | − | − | − | − |
B. licheniformis +ve control | + | + | + | − |
B. pumilus -ve control | − | − | − | − |
Farm 1, Winter 1 | + | + | + | − |
Farm 1, Winter 3 | + | + | + | − |
Farm 1, Winter 4 | + | + | + | − |
Farm 1, Winter 8 | + | + | + | − |
Farm 1, Winter 9 | + | + | + | − |
Farm 1, Winter 19 | + | + | + | − |
Farm 1, Winter 25 | + | + | + | − |
Farm 1, Winter 26 | + | + | + | − |
Farm 1, Winter 27 | + | + | + | − |
B. licheniformis +ve control | + | + | + | − |
Farm 2, Summer 1 | + | + | + | − |
Farm 2, Summer 2 | + | + | + | − |
Farm 2, Winter 2 | + | + | + | − |
Farm 2, Winter 9 (B. pumilus) | − | − | − | − |
B. licheniformis +ve control | + | + | + | − |
B. pumilus -ve control | − | − | − | − |
Farm 3, Winter 1 | + | + | + | − |
Farm 3, Winter 9 | + | + | + | − |
Farm 3, Winter 10 | + | + | + | − |
Farm 3, Winter 3 (B. pumilus) | − | − | − | − |
B. licheniformis +ve control | + | + | + | − |
B. pumilus -ve control | − | − | − | − |
Farm 4, Summer 1 | + | + | + | − |
Farm 4, Summer 5 | + | + | + | − |
Farm 4, Summer 29 | − | − | − | − |
B. licheniformis +ve control | + | + | + | − |
Farm 4, Winter 1 | + | + | + | − |
Farm 4, Winter 4 | + | + | + | − |
Farm 4, Winter 5 | + | + | + | − |
Farm 4, Winter 15 | + | + | + | − |
Farm 4, Winter 31 | + | + | + | − |
B. licheniformis +ve control | + | + | + | − |
Isolates | ||||
---|---|---|---|---|
Toxin Genes | B. cereus NCTC 11143 (Positive Control) | Farm 1 Winter 11 (B. mycoides) | Farm 1 Winter 12 (B. mycoides) | Farm 3 Winter 17 (B. cereus) |
hblA | + | + | + | − |
hblC | + | + | + | − |
hblD | + | − | − | + |
nheA | + | + | + | − |
nheB | + | + | + | − |
nheC | + | − | − | − |
entFM | + | − | − | + |
cytK1 | + | − | − | − |
cesB | + | − | − | − |
Isolate | PCR | ELISA | Toxinotype | |||||
---|---|---|---|---|---|---|---|---|
cpa | cpb | etx | iap and ibp | Alpha | Beta | Epsilon | ||
Farm 3, Winter 1 | + | − | + | − | ++ | − | ++ | D |
Farm 3, Winter 2 | + | − | + | − | ++ | − | ++ | D |
Farm 3, Winter 3 | + | − | − | − | ++ | − | A | |
Farm 3, Winter 4 | + | − | + | − | ++ | − | D# | |
Farm 3, Winter 5 | + | − | + | − | ++ | − | ++ | D |
Farm 3, Winter 6 | + | − | − | − | ++ | − | A | |
Farm 3, Winter 7 | + | − | − | − | ++ | − | A | |
Farm 3, Winter 8 | + | − | − | − | ++ | − | A | |
Farm 3, Winter 9 | + | − | − | − | ++ | − | A | |
Farm 3, Winter 10 | + | − | − | − | ++ | − | A | |
Farm 3, Winter 11 | + | − | − | − | ++ | − | A | |
Farm 3, Winter 12 | + | − | − | − | ++ | − | − | A |
Farm 3, Winter 13 | + | − | − | − | ++ | − | − | A |
Farm 3, Winter 14 | + | − | − | − | ++ | − | − | A |
Farm 3, Winter 15 | + | − | − | − | ++ | − | − | A |
Farm 4, Winter 1 | + | − | − | − | ++ | − | A | |
Farm 4, Winter 2 | + | − | − | − | ++ | − | A | |
Farm 4, Winter 3 | + | − | − | − | ++ | − | A | |
Farm 4, Winter 4 | + | − | − | − | ++ | − | A | |
Farm 4, Winter 5 | + | − | − | − | ++ | − | A | |
Farm 4, Winter 6 | + | − | − | − | ++ | − | A | |
Farm 4, Winter 7 | + | − | − | − | ++ | − | A | |
Farm 4, Winter 8 | + | − | − | − | ++ | − | A | |
Farm 4, Winter 9 | + | − | − | − | ++ | − | A | |
Farm 4, Winter 10 | + | + | + | − | ++ | ++ | ++ | B |
Farm 4, Winter 11 | + | + | + | − | ++ | ++ | ++ | B |
Farm 4, Winter 12 | + | + | + | − | ++ | ++ | ++ | B |
Spoilage Activity | ||||
---|---|---|---|---|
Farm, Season | Isolate Number | Proteolytic | Lipolytic | Bacteria |
Farm 1, Summer | 1 | B. licheniformis | ||
2 | B. pumilus | |||
4 | P. marinisediminis | |||
5 | B. licheniformis | |||
7 | B. licheniformis | |||
8 | B. licheniformis | |||
9 | B. licheniformis | |||
11 | B. licheniformis | |||
12 | B. licheniformis | |||
13 | B. licheniformis | |||
Farm 1, Winter | 1 | B. licheniformis | ||
2 | P. xylanexedens | |||
3 | B. licheniformis | |||
4 | B. licheniformis | |||
6 | B. clausii | |||
8 | B. licheniformis | |||
9 | B. licheniformis | |||
10 | P. peoriae | |||
12 | B. mycoides | |||
14 | B. altitudinis | |||
17 | B. circulans | |||
19 | B. licheniformis | |||
24 | S. silvestris | |||
25 | B. licheniformis | |||
26 | B. licheniformis | |||
27 | B. licheniformis | |||
28 | B. circulans | |||
Farm 2, Summer | 1 | B. licheniformis | ||
2 | B. licheniformis | |||
Farm 2, Winter | 1 | B. pumilus | ||
2 | B. licheniformis | |||
8 | B. kochii | |||
9 | B. pumilus | |||
10 | S. silvestris | |||
11 | B. rigui | |||
12 | NT | NT | S. luteola | |
13 | P. polymyxa | |||
Farm 3, Winter | 1 | B. licheniformis | ||
2 | B. clausii | |||
3 | B. pumilus | |||
6 | B. pumilus | |||
8 | Ornithinibacillus | |||
9 | B. licheniformis | |||
10 | B. licheniformis | |||
11 | B. licheniformis | |||
12 | B. licheniformis | |||
13 | B. pumilus | |||
17 | B. cereus | |||
22 | B. clausii | |||
23 | P. amylolyticus | |||
Farm 4, Summer | 1 | B. licheniformis | ||
5 | B. licheniformis | |||
6 | B. subtilis | |||
17 | B. clausii | |||
22 | P. timonensis | |||
25 | L. fusiformis | |||
27 | B. kochii | |||
29 | B. licheniformis | |||
Farm 4, Winter | 1 | B. licheniformis | ||
3 | B. altitudinis | |||
4 | B. licheniformis | |||
5 | B. licheniformis | |||
9 | B. simplex | |||
15 | B. licheniformis | |||
22 | B. kochii | |||
24 | L. fusiformis | |||
25 | P. cookii | |||
26 | B. clausii | |||
29 | B. simplex | |||
30 | B. pocheonensis | |||
31 | B. licheniformis | |||
32 | P. lactis |
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Gupta, T.B.; Brightwell, G. Identification and Characterisation of Spore-Forming Bacteria in Bovine Raw Milk Collected from Four Dairy Farms in New Zealand. Dairy 2023, 4, 650-671. https://doi.org/10.3390/dairy4040045
Gupta TB, Brightwell G. Identification and Characterisation of Spore-Forming Bacteria in Bovine Raw Milk Collected from Four Dairy Farms in New Zealand. Dairy. 2023; 4(4):650-671. https://doi.org/10.3390/dairy4040045
Chicago/Turabian StyleGupta, Tanushree B., and Gale Brightwell. 2023. "Identification and Characterisation of Spore-Forming Bacteria in Bovine Raw Milk Collected from Four Dairy Farms in New Zealand" Dairy 4, no. 4: 650-671. https://doi.org/10.3390/dairy4040045
APA StyleGupta, T. B., & Brightwell, G. (2023). Identification and Characterisation of Spore-Forming Bacteria in Bovine Raw Milk Collected from Four Dairy Farms in New Zealand. Dairy, 4(4), 650-671. https://doi.org/10.3390/dairy4040045