Impact of the Technical Snow Production Process on Bacterial Community Composition, Antibacterial Resistance Genes, and Antibiotic Input—A Dual Effect of the Inevitable
Abstract
1. Introduction
2. Results and Discussion
2.1. Culture-Based Assessment of Bacteriological Contamination
2.2. Concentration of Antimicrobial Agents
2.3. Detection and Prevalence of Antibiotic Resistance Genes (ARGs)
2.4. Bacterial Community Diversity and Composition
2.5. Multivariate Analysis of Data
3. Materials and Methods
3.1. Sampling Sites
Code | Height Above Sea Level [m a.s.l] | Number of Inhabitants | Anthropogenic Pressure Description | Technical Reservoir [yes/no] | Sample Description and Code |
---|---|---|---|---|---|
BDF | 850 | 540 | upstream of a small village next to the Tatra National Park, upstream of wastewater discharge sites | yes | river water (BDF_W) |
storage reservoir (BDF_R) | |||||
snowmelt water (BDF_S) | |||||
B3 | 760 | 950 | upstream of a small village next to the Tatra National Park and the Polish/Slovakian border | no | river water (B3_W) |
snowmelt water (B3_S) | |||||
B1 | 700 | 2300 | center of a popular tourist resort, ca. 3 km downstream of a WWTP | yes | river water (B1_W) |
storage reservoir (B1_R) | |||||
snowmelt water (B1_S) | |||||
R | 315 | 17,500 | center of a medium-sized town, downstream of several ski resorts, ca. 5 km downstream of a hospital, ca. 10 km of a WWTP | no | river water (R_W) |
snowmelt water (R_S) | |||||
BDZ | 750 | 25,000 | center of a popular tourist resort, ca. 3 km downstream of a WWTP, ca. 2 km downstream of a hospital | no | river water (BDZ_W) snowmelt water (BDZ_S) |
3.2. Sample Collection
3.3. Culture-Based Microbiological Analysis of Samples
3.4. Determining the Presence and Concentration of Antimicrobial Agents in Water and Snowmelt Samples
3.5. PCR Determination of Genetic Antimicrobial Resistance Determinants in Total Genomic DNA
3.6. Illumina Sequencing of V3-V4 16S rRNA Amplicon
3.7. Statistical Analyses
4. Conclusions
- ○
- The numbers of culturable bacteria drop sharply during the technical snowmaking process; thus, even if the snow is produced from water severely contaminated by wastewater, the resulting snow does not seem to pose a serious threat to human health.
- ○
- The presence and concentration of antimicrobial agents in water and the produced snow is strongly affected by the proximity of point sources of pollution, such as WWTPs and hospitals.
- ○
- As many as nine antibiotic resistance genes (ARGs) were detected at the examined sites, and their prevalence was strongly affected by the close vicinity of wastewater discharge sites and hospitals. The presence and concentration of antimicrobial agents is also associated with the occurrence of ARGs.
- ○
- Importantly, the prevalence of most ARGs decreased during technical snowmaking. This was more evident within ski stations where water is stored in technical reservoirs prior to snowmaking. On the other hand, biofilm formation and its further detachment may contribute to reduced removal efficiency of ARGs.
- ○
- Finally, the NGS-based analysis of bacterial community composition evidently indicates its changes through the process of technical snowmaking, which may be due to the survivability of certain strains in freezing temperatures or the inhibitory effect of antimicrobial agents that enter the technical snow.
- ○
- The presence and concentration of antimicrobials in water seem to be the most significant factors affecting the changes in bacterial community composition. Therefore, measures should be taken to reduce their spread within the aquatic environment.
- ○
- Among the measures that a ski station management could implement, one can mention the construction of technical reservoirs. These appear to effectively eliminate pollutants and micropollutants from the resource water. In addition, ensuring the regular and frequent cleaning and maintenance of snowmaking devices is advisable.
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Code | Sample Description and Code | E. coli | E. faecalis/E. faecium | Salmonella | Coagulase-Positive Staphylococci |
---|---|---|---|---|---|
CFU/100 mL | CFU/mL | ||||
BDF | river water (BDF_W) | 1 | 4 | 1 | 0 |
storage reservoir (BDF_R) | 0 | 5 | 0 | 350 | |
snowmelt water (BDF_S) | 1 | 7 | 1 | 0 | |
B3 | river water (B3_W) | 119 | 45 | 0 | 2 |
snowmelt water (B3_S) | 0 | 26 | 0 | 2 | |
B1 | river water (B1_W) | 186 | 94 | 0 | 0 |
storage reservoir (B1_R) | 14 | 13 | 0 | 0 | |
snowmelt water (B1_S) | 2 | 7 | 0 | 12 | |
R | river water (R_W) | 298 | 45 | 56 | 328 |
snowmelt water (R_S) | 87 | 26 | 5 | 232 | |
BDZ | river water (BDZ_W) | 224,067 | 273,641 | 10,785 | 8667 |
snowmelt water (BDZ_S) | 153 | 286 | 4 | 190 |
Chemical Group | Antibiotic | BDF_W | BDF_R | BDF_S | B1_W | B1_R | B1_S | R_W | R_S | B3_W | B3_S | BDZ_W | BDZ_S | Frequency of Detection (% of All Samples) | Total Concentration of Antibiotics in All Samples (ng/L) |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
2nd gen. cephalosporins | cefoxitin | 0.00 | 0.00 | 112.59 | 0.00 | 0.00 | 73.47 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 9.7 | 519.05 |
fluoroquinolones | ciprofloxacin | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 65.20 | 24.31 | 0.00 | 0.00 | 0.00 | 15.37 | 9.7 | 314.65 |
enrofloxacin | 0.00 | 0.00 | 0.00 | 1.67 | 6.09 | 3.04 | 626.28 | 34.95 | 3.38 | 0.95 | 2.94 | 0.00 | 3.2 | 204.78 | |
ofloxacin | 0.00 | 0.00 | 0.00 | 0.49 | 0.00 | 0.00 | 95.64 | 28.39 | 0.00 | 0.00 | 7.00 | 6.29 | 29.0 | 2030.53 | |
lincosamids | clindamycin | 0.51 | 0.00 | 0.00 | 1.83 | <LOQ ** | 2.47 | 37.66 | 9.96 | 5.80 | 0.00 | 15.59 | 11.18 | 29.0 | 3.71 |
macrolides | erythromycin | 0.10 | 0.00 | 0.00 | 0.07 | 0.00 | 0.05 | 0.30 | 0.24 | 0.00 | 0.00 | 0.30 | 0.20 | 71.0 | 251.15 |
tylosin | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 56.59 | 10.23 | 0.00 | 0.00 | 0.00 | 0.00 | 22.6 | 64.45 | |
tetracyclines | doxycycline | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 68.26 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 16.1 | 413.43 |
oxytetracycline | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 13.47 | 0.00 | 0.00 | 0.00 | 0.00 | 61.36 | 6.5 | 224.46 | |
tetracycline | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 9.23 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 35.5 | 214.61 | |
sulphonamids | sulfamethoxazole | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 1.56 | 20.90 | 4.69 | 1.48 | 0.00 | 34.05 | 8.83 | 3.2 | 27.70 |
antifolates | trimethoprim | 0.00 | 0.00 | 0.00 | 1.10 | 0.00 | 0.18 | 38.57 | 7.70 | 2.59 | 0.00 | 8.61 | 4.93 | 35.5 | 188.63 |
glycopeptides | vancomycin | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 142.66 | 21.84 | 0.00 | 0.00 | 4.23 | 0.00 | 9.7 | 506.19 |
oxazolidinones | linezolid | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 1.33 | 17.40 | 1.48 | 1.63 | 0.00 | 3.99 | 1.55 | 6.5 | 200.44 |
number of antibiotics detected | 2 | 0 | 1 | 5 | 2 | 7 | 13 | 10 | 5 | 1 | 8 | 8 | |||
total concentration of antibiotics | 1.21 | 0 | 225.19 | 15.45 | 12.17 | 328.40 | 3559.06 | 431.37 | 29.77 | 1.90 | 230.14 | 329.10 |
Site | Beta-Lactamases | Altered Penicillin-Binding Protein (PBP2a) | Erythromycin Esterase | Macrolide Ribosomal Methylase | Aminoglycoside 3’-phosphotransferase | Tetracycline Efflux Protein | Dihydropteroate Synthase | ||
---|---|---|---|---|---|---|---|---|---|
blaTEM | blaCTX-M | blaCTX-M3 | mecA | ereA | ermB | strA | tetK | sulIII | |
BDF | 16.7 | 16.7 | 0 | 0 | 0 | 0 | 16.7 | 50.0 | 0 |
B3 | 50.0 | 25.0 | 0 | 0 | 0 | 0 | 0 | 50.0 | 0 |
B1 | 12.5 | 12.5 | 0 | 0 | 0 | 12.5 | 37.5 | 75 | 0 |
R | 66.7 | 16.7 | 0 | 16.7 | 16.7 | 0 | 66.7 | 33.3 | 16.7 |
BDZ | 100 | 33.3 | 16.7 | 0 | 83.3 | 100 | 100 | 66.7 | 16.7 |
total share | 46.7 | 20.0 | 3.3 | 3.3 | 20.0 | 23.3 | 46.7 | 56.7 | 6.7 |
No. | Resistance Mechanism | Gene | Primer | Sequence (5′-3′) | Annealing Temp. (°C) | Product Length (bp) | Reference |
---|---|---|---|---|---|---|---|
1. | Extended-spectrum beta-lactamases (ESBL) | blaTEM | blaTEM-F | ATTCTTGAAGACGAAAGGGC ACGCTCAGTGGAACGAAAAC | 60 | 1150 | [67] |
blaTEM-R | |||||||
2. | blaCTX-M | blaCTX-M-F | CGATGTGCAGTACCAGTAA TTAGTGACCAGAATCAGCGG | 55 | 585 | [68] | |
blaCTX-M-R | |||||||
3. | blaCTX-M3 | blaCTX-M3-F | GTTACAATGTGTGAGAAGCAG CCGTTTCCGCTATTACAAAC | 50 | 1049 | [69] | |
blaCTX-M3-R | |||||||
4. | blaCTX-M9 | blaCTX-M9-F | GTGACAAAGAGAGTGCAACGG ATGATTCTCGCCGCTGAAGCC | 54 | 856 | [70] | |
blaCTX-M9-R | |||||||
5. | blaSHV | blaSHV-F | CACTCAAGGATGTATTGTG TTAGCGTTGCCAGTGCTCG | 52 | 885 | [67] | |
blaSHV-R | |||||||
6. | blaOXA-1 | blaOXA-1-F | ACACAATACATATCAACTTCGC AGTGTGTTTAGAATGGTGATC | 61 | 813 | [67] | |
blaOXA-1-R | |||||||
7. | Carbapenemases class D | blaOXA-48 | blaOXA-48-F | GCTTGATCGCCCTCGATT GATTTGCTCCGTGGCCGAAA | 60 | 281 | [71] |
blaOXA-48-R | |||||||
8. | Carbapenemases class A | blaKPC | blaKPC-F | TGTTGCTGAAGGAGTTGGGC ACGACGGCATAGTCATTTGC | 57 | 340 | [71] |
blaKPC-R | |||||||
9. | Carbapenemases class B | blaIMP | blaIMP-F | TTGACACTCCATTTACAG GATCGAGAATTAAGCCACCC | 56 | 139 | [71] |
blaIMP-R | |||||||
10. | blaVIM | blaVIM-F | GATGGTGTTTGGTCGCATA CGAATGCGCAGCACCAG | 60 | 390 | [71] | |
blaVIM-R | |||||||
11. | blaNDM | blaNDM-F | GGTTTGGCGATCTGGTTTTC CGGAATGGCTCATCACGATC | 60 | 621 | [72] | |
blaNDM-R | |||||||
12. | Methicillin-resistance | mecA | mecA-F | GTAGAAAATGACTGAACGTCCGATAA CCAATTCCACATTGTTTCGGTCTAA | 55 | 310 | [73] |
mecA-R | |||||||
13. | Macrolide–lincosamide–streptogramin B (MLSb) resistance genes | ereA | ereA-F ereA-R | AACACCCTGAACCCAAGGGACG CTTCACATCCGGATTCGCTCGA | 57 | 420 | [74] |
14. | ereB | ereB-F ereB-R | AGAAATGGAGGTTCATACTTACCA CATATAATCATCACCAATGGCA | 52 | 546 | [74] | |
15. | ermA | ermA-F ermA-R | TCTAAAAAGCATGTAAAAGAA CTTCGATAGTTTATTAATATTAGT | 52 | 645 | [74] | |
16. | ermB | ermB-F ermB-R | GAAAAGGTACTCAACCAAATA AGTAACGGTACTTAAATTGTTTAC | 55 | 639 | [74] | |
17. | msrA | msrA-F msrA-R | GGCACAATAAGAGTGTTTAAAGG AAGTTATATCATGAATAGATTGTCCTGTT | 50 | 940 | [75] | |
18. | msrB | msrB-F msrB-R | TATGATATCCATAATAATTATCCAATC AAGTTATATCATGAATAGATTGTCCTGTT | 50 | 595 | [75] | |
19. | mphA | mphA-F mphA-R | AACTGTACGCACTTGC GGTACTCTTCGTTACC | 50 | 837 | [74] | |
20. | lnuA | lnuA-F lnuA-R | GGTGGCTGGGGGGTAGATGTATTAACTGG GCTTCTTTTGAAATACATGGTATTTTTCGATC | 57 | 323 | [75] | |
21. | vatA | vatA-F vatA-R | CAATGACCATGGACCTGATC CTTCAGCATTTCGATATCTC | 52 | 619 | [75] | |
22. | vatB | vatB-F vatB -R | CCCTGATCCAAATAGCATATATCC CTAAATCAGAGCTACAAAGT | 52 | 602 | [75] | |
23. | vga | vga-F vga-R | CCAGAACTGCTATTAGCAGATGAA AAGTTCGTTTCTCTTTTCGACG | 54 | 470 | [75] | |
24. | vgb | vgb-F vgb-R | ACTAACCAAGATACAGGACC TTATTGCTTGTCAGCCTTCC | 53 | 734 | [75] | |
25. | Streptomycin resistance | strA | strA-F strA-R | TCAATCCCGACTTCTTACCG CACCATGGCAAACAACCATA | 52 | 126 | [76] |
26. | Trimetophrim resistance | dfrA12 | dfrA12-F dfrA12-R | TTTATCTCGTTGCTGCGATG AGGCTTGCCGATAGACTCAA | 50 | 155 | [77] |
27. | Aminoglycoside resistance | aac(6′)/aph(2′) | aac(6′)/aph(2′′)-F aac(6′)/aph(2′′)-R | CAGAGCCTTGGGAAGATGAAG CCTCGTGTAATTCATGTTCTGGC | 55 | 348 | [78] |
28. | Tetracyclines resistance | tetK | tetK-F tetK-R | TCGATAGGAACAGCAGTA CAGCAGATCCTACTCCTT | 55 | 169 | [79] |
29. | Sulfonamides resistance | sulIII | sulIII-F sulIII-R | ACCACCGATAGTTTTTCCGA TGCCTTTTTCTTTTAAAGCC | 62 | 199 | [77] |
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Stankiewicz, K.; Bulanda, K.; Prajsnar, J.; Lenart-Boroń, A. Impact of the Technical Snow Production Process on Bacterial Community Composition, Antibacterial Resistance Genes, and Antibiotic Input—A Dual Effect of the Inevitable. Int. J. Mol. Sci. 2025, 26, 2771. https://doi.org/10.3390/ijms26062771
Stankiewicz K, Bulanda K, Prajsnar J, Lenart-Boroń A. Impact of the Technical Snow Production Process on Bacterial Community Composition, Antibacterial Resistance Genes, and Antibiotic Input—A Dual Effect of the Inevitable. International Journal of Molecular Sciences. 2025; 26(6):2771. https://doi.org/10.3390/ijms26062771
Chicago/Turabian StyleStankiewicz, Klaudia, Klaudia Bulanda, Justyna Prajsnar, and Anna Lenart-Boroń. 2025. "Impact of the Technical Snow Production Process on Bacterial Community Composition, Antibacterial Resistance Genes, and Antibiotic Input—A Dual Effect of the Inevitable" International Journal of Molecular Sciences 26, no. 6: 2771. https://doi.org/10.3390/ijms26062771
APA StyleStankiewicz, K., Bulanda, K., Prajsnar, J., & Lenart-Boroń, A. (2025). Impact of the Technical Snow Production Process on Bacterial Community Composition, Antibacterial Resistance Genes, and Antibiotic Input—A Dual Effect of the Inevitable. International Journal of Molecular Sciences, 26(6), 2771. https://doi.org/10.3390/ijms26062771