Microbial Source Tracking as a Method of Determination of Beach Sand Contamination
Abstract
:1. Introduction
The Case under Study
2. Materials and Methods
2.1. Collection and Preservation of the Sand Samples
2.2. Microbiological Parameters
2.3. DNA Extraction from Sand Samples
2.4. Microbial Source Tracking (MST) Analyses
2.4.1. Primers
2.4.2. DNA Extraction for MST Validation
2.4.3. PCR Detection 1—Fresh Coastal Sand Samples
2.4.4. PCR Detection 2—Fecal Samples for MST Validation and Frozen Coastal Sand Samples
3. Results
3.1. Management and Remediation Measures
3.2. MST Markers Validation
3.3. Microbiological and MST Results
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Target | Primers’ Names | Sequence (5′–3′) | Conventional PCR Annealing Temperature | Tested by Probe-Based qPCR | References |
---|---|---|---|---|---|
Humans | HF183F BacR287 | F: ATCATGAGTTCACATGTCCG R: CTTCCTCTCAGAACCCCTATCC P: FAM–CTAATGGAACGCATCCC–MGBEQ | 53 °C | YES | [51] |
Dogs | DF113F DF472R | F: ATCTCAAGAGCACATGCAA R: AATAAATCCGGATAACGCTC | 53 °C | NO | [21] |
Seagulls | Gull-2F Gull-2R | F: TGCATCGACCTAAAGTTTTGAG R: GTCAAAGAGCGAGCAGTTACTA | 53 °C | NO | [52] |
Ruminants | RUM_CF128F RUM_Bac708R | F: CCAACYTTCCCGWTACTC R: CAATCGGAGTTCTTCGTG | 60 °C | NO | [46] |
Cows | CowM2F CowM2R | F: CGGCCAAATACTCCTGATCGT R: GCTTGTTGCGTTCCTTGAGATAAT P: FAM–AGGCACCTATGTCCTTTACCT CATCAACTACAGACA–MGBEQ | ND | YES | [53] |
Cows | CowM3F CowM3R | F: CCTCTAATGGAAAATGGATGGTATCT R: CCATACTTCGCCTGCTAATACCTT | 53 °C | NO | [53] |
Pigs | Bac41F Bac163R | F: GCATGAATTTAGCTTGCTAAATTTGAT R: ACCTCATACGGTATTAATCCGC | 60 °C | NO | [22] |
Sampling Date | 21 August 2019 | 27 August 2019 | 3 September 2019 | 10 September 2019 | |||||
---|---|---|---|---|---|---|---|---|---|
Sampling Sites | Fecal Indicator Bacteria Results | MST Results | Fecal Indicator Bacteria Results | ||||||
Humans | Dogs | Seagulls | Ruminants | Bovine | |||||
Site 1 | Coliform bacteria, 1 MPN/g Escherichia coli, 1 MPN/g Enterococcus spp., <1 MPN/g | NEG | NEG | NEG | NEG | ND | Coliform bacteria, <1 MPN/g Escherichia coli, <1 MPN/g Enterococcus spp., <1 MPN/g | ||
Site 2 | Coliform bacteria, 201 MPN/g Escherichia coli, 84 MPN/g Enterococcus spp., 201 MPN/g | NEG | NEG | NEG | NEG | NEG | Coliform bacteria, 4 MPN/g Escherichia coli, <1 MPN/g Enterococcus spp., <1 MPN/g | Coliform bacteria, 102 MPN/g Escherichia coli, <1 MPN/g Enterococcus spp., <1 MPN/g | Coliform bacteria, <1 MPN/g Escherichia coli, <1 MPN/g Enterococcus spp., <1 MPN/g |
Site 3 | Coliform bacteria, >201 MPN/g Escherichia coli, >201 MPN/g Enterococcus spp., 201 MPN/g | NEG | NEG | NEG | NEG | ND | Coliform bacteria, 4 MPN/g Escherichia coli, <1 MPN/g Enterococcus spp., <1 MPN/g | Coliform bacteria, <1 MPN/g Escherichia coli, <1 MPN/g Enterococcus spp., <1 MPN/g | |
Site 4 | Coliform bacteria, 14 MPN/g Escherichia coli, 1 MPN/g Enterococcus spp., 10 MPN/g | NEG | POS | NEG | NEG | NEG | Coliform bacteria, 9 MPN/g Escherichia coli, <1 MPN/g Enterococcus spp., 9 MPN/g | Coliform bacteria, <1 MPN/g Escherichia coli, <1 MPN/g Enterococcus spp., <1 MPN/g | |
Site 5 | Coliform bacteria, >201 MPN/g Escherichia coli, 110 MPN/g Enterococcus spp., 74 MPN/g | NEG | NEG | POS | NEG | ND | Coliform bacteria, <1 MPN/g Escherichia coli, <1 MPN/g Enterococcus spp., <1 MPN/g | Coliform bacteria, <1 MPN/g Escherichia coli, <1 MPN/g Enterococcus spp., <1 MPN/g | |
Site 6 | Coliform bacteria, 166 MPN/g Escherichia coli, 63 MPN/g Enterococcus spp., 51 MPN/g | NEG | NEG | NEG | NEG | ND | Coliform bacteria, <1 MPN/g Escherichia coli, <1 MPN/g Enterococcus spp., <1 MPN/g | ||
Site 7 | Coliform bacteria, 5 MPN/g Escherichia coli, 1 MPN/g Enterococcus spp., 130 MPN/g | NEG | NEG | NEG | NEG | NEG | Coliform bacteria, <1 MPN/g Escherichia coli, <1 MPN/g Enterococcus spp., <1 MPN/g | ||
Site 8 | Coliform bacteria, >201 MPN/g Escherichia coli, 12 MPN/g Enterococcus spp., 24 MPN/g | NEG | NEG | NEG | POS | NEG | Coliform bacteria, 1 MPN/g Escherichia coli, <1 MPN/g Enterococcus spp., 1 MPN/g | ||
Site 9 | Coliform bacteria, >201 MPN/g Escherichia coli, 28 MPN/g Enterococcus spp., 28 MPN/g | NEG | NEG | NEG | POS | NEG | Coliform bacteria, <1 MPN/g Escherichia coli, <1 MPN/g Enterococcus spp., <1 MPN/g | Coliform bacteria, <1 MPN/g Escherichia coli, <1 MPN/g Enterococcus spp., <1 MPN/g | Coliform bacteria, <1 MPN/g Escherichia coli, <1 MPN/g Enterococcus spp., <1 MPN/g |
Site 10 | Coliform bacteria, 201 MPN/g Escherichia coli, 13 MPN/g Enterococcus spp., 4 MPN/g | NEG | NEG | NEG | NEG | ND | Coliform bacteria, 2 MPN/g Escherichia coli, 1 MPN/g Enterococcus spp., <1 MPN/g | Coliform bacteria, <1 MPN/g Escherichia coli, <1 MPN/g Enterococcus spp., <1 MPN/g | |
Site 11 | Coliform bacteria, <1 MPN/g Escherichia coli, <1 MPN/g Enterococcus spp., <1 MPN/g |
Target | Primers’ Names | Humans | Domestic Animals | Livestock | Birds | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Cat | Dog | Donkey | Horse | Cow | Goat | Sheep | Pig | Canaries | Seagull | Chicken | Duck | Turkey | |||
Humans | HF183F BacR287 | ✓ | ND | ✗ | ✗ | ✗ | ✗ | ✗ | ✗ | ✗ | ND | ✗ | ✗ | ✗ | ✗ |
Dogs | DF113F DF472R | ✗ | ✗ | ✓ | ND | ✗ | ✗ | ✗ | ✗ | ✗ | ✗ | ND | ✗ | ND | ND |
Seagulls | Gull-2F Gull-2R | ✗ | ND | ✗ | ND | ✗ | ✗ | ✗ | ✗ | ✗ | ✗ | ✓ | ✗ | ✗ | ✗ |
Ruminants | RUM_CF128F RUM_Bac708R | ✗ | ND | ✗ | ✗ | ⚠ | ✓ | ✓ | ✓ | ✗ | ND | ND | ✗ | ND | ND |
Cows | CowM3F CowM3R | ✗ | ND | ✗ | ✗ | ✗ | ✓ | ✗ | ✗ | ✗ | ND | ND | ND | ✗ | ND |
Pigs | Bac41F Bac163R | ✗ | ND | ✗ | ✗ | ✗ | ✗ | ✗ | ✗ | ✓ | ND | ND | ✗ | ND | ND |
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Valério, E.; Santos, M.L.; Teixeira, P.; Matias, R.; Mendonça, J.; Ahmed, W.; Brandão, J. Microbial Source Tracking as a Method of Determination of Beach Sand Contamination. Int. J. Environ. Res. Public Health 2022, 19, 7934. https://doi.org/10.3390/ijerph19137934
Valério E, Santos ML, Teixeira P, Matias R, Mendonça J, Ahmed W, Brandão J. Microbial Source Tracking as a Method of Determination of Beach Sand Contamination. International Journal of Environmental Research and Public Health. 2022; 19(13):7934. https://doi.org/10.3390/ijerph19137934
Chicago/Turabian StyleValério, Elisabete, Maria Leonor Santos, Pedro Teixeira, Ricardo Matias, João Mendonça, Warish Ahmed, and João Brandão. 2022. "Microbial Source Tracking as a Method of Determination of Beach Sand Contamination" International Journal of Environmental Research and Public Health 19, no. 13: 7934. https://doi.org/10.3390/ijerph19137934