Combined effects of ciprofloxacin and microplastics on alpine spring water microbiota: evidence from glacier-fed microcosm experiments

Abstract

Introduction: Emerging contaminants such as microplastics (MPs) and antibiotics pose increasing environmental and public health risks due to their persistence and incomplete removal by wastewater treatment processes. MPs can act as vectors for antibiotics, facilitating their environmental spreading and supporting biofilm formation, which can enhance horizontal gene transfer and antibiotic resistance. This study investigates the combined effects of ciprofloxacin (CIP) and polyethylene terephthalate (PET) MPs on microbiota in alpine spring water (SW) sourced from a rock glacier.

Methods: Four experimental scenarios (Control, CIP, PET, CIP + PET) were established to assess the sorption dynamics of CIP onto PET particles and the consequent microbial responses. A multidisciplinary analytical approach combining ultra-performance liquid chromatography, microscopy, quantitative PCR, and metabarcoding was applied.

Results: CIP exhibited progressive sorption onto PET, accompanied by a time-dependent increase in biofilm formation, most pronounced in the CIP + PET condition. qPCR revealed elevated copy numbers of resistance genes qnrA and qnrB in CIP + PET, suggesting synergistic effects between antibiotics and MPs in promoting resistance. CIP was the dominant driver of microbial compositional shifts, favoring known CIP-degrading taxa. A shared core microbiome of 216 amplicon sequence variants was detected across all conditions, but specific taxa were differentially enriched under varying exposures. The combined CIP + PET test induced the strongest community shifts, while CIP alone shared fewer taxa with controls, indicating selective pressure for resistant microorganisms like Achromobacter. PET MPs also shaped distinct microbial assemblages, possibly by offering niches favoring biofilm-associated genera such as Luteolibacter. Biodiversity metrics showed highest richness and evenness in CIP-free conditions (Control and PET), while CIP significantly reduced alpha diversity, favoring resistant taxa, as confirmed by NMDS and lower Shannon and Simpson indices. Effects of MPs were still noticeable.

Conclusion: These findings demonstrate the disruptive effects of CIP on alpine freshwater microbial communities and highlight the additional, though more moderate, influence of MPs. The combined presence of MPs and antibiotics may exacerbate resistance spreading by enhancing persistence and providing favorable conditions for resistant biofilms. A mechanistic understanding of these interactions is essential for accurate risk assessment and the development of effective mitigation strategies in alpine and other vulnerable freshwater ecosystems.

Keywords: alpine ecosystem; antibiotic resistance; emerging contaminants; freshwater; microplastics pollution; polyethylene terephthalate.

Figure 1

Minimum, maximum and average Ra values estimated for each MP-CIP pair.

Figure 2

Time profiles of CIP concentrations in aqueous phases of B (A) and D (B) conditions and in PET MPs (C) in D conditions and CIP mass balance in D conditions (D).

Figure 3

Venn diagram showing the number of shared and unique amplicon sequence variants (ASVs) among the four experimental conditions (A = CTRL; B = CIP; C = PET; D = CIP + PET). The overlaps represent ASVs detected in more than one condition, while non-overlapping areas indicate condition-specific taxa.

Figure 4

Taxonomic composition of microbial communities at the genus level (20 most prevalent genera) across the four experimental treatments (A: CTRL, B: CIP, C: PET, D: CIP + PET) and four incubation time point (T1: 10 days; T2: 20 days; T3: 40 days; T4: 60 days).

Figure 5

Non-metric multidimensional scaling (NMDS) plot based on Bray–Curtis dissimilarity, illustrating differences in microbial community composition across treatments (A: CTRL, B: CIP, C: PET, D: CIP + PET), timepoints (T1: 10 days; T2: 20 days; T3: 40 days; T4: 60 days) and replicates (a, b). Each point represents a microbial community from a sample, with colors indicating treatment groups. Clustering of samples reflects compositional similarity, with tighter groupings indicating higher similarity in community structure.

Figure 6

Predicted functional pathways related to antibiotic resistance, biofilm formation, and transporters. (A) Donut chart showing the relative abundance of selected KEGG pathways predicted across all samples based on 16S rRNA gene data using the Tax4Fun pipeline. Functional categories include antibiotic biosynthesis, various antibiotic resistance mechanisms (e.g., β-lactam, vancomycin, CAMP), biofilm formation in specific bacteria (e.g., Pseudomonas aeruginosa, Escherichia coli, Vibrio cholerae), and membrane transporters (ABC transporters). (B) Stacked barplot displaying the distribution of these pathways across individual treatments (A: CTRL, B: CIP, C: PET, D: CIP + PET).

Figure 7

Quantification of CIP resistance genes across four experimental conditions (CTRL, CIP, PET, CIP + PET) and four incubation time points (10 days, 20 days, 40 days and 60 days). Panels show relative abundances of (A) qnrA, (B) qnrB, (C) qnrC, and (D) qnrS, highlighting the temporal dynamics and treatment-specific variations in the presence of plasmid-mediated quinolone resistance (PMQR) genes.

Figure 8

(A) Ciprofloxacin sorption rates and relative abundance of resistance genes (qnrA, qnrB, qnrC, qnrS) in sample D (CIP + PET) over 10, 20, 40, and 60 days. Gene abundances are log-transformed. (B) Circular phylogenetic tree of bacterial ASVs enriched in sample D across three incubation times (T2 = 20 days, T3 = 40 days, T4 = 60 days) correlating CIP sorption rates. The outer-colored bars represent the log-transformed relative abundance of each phylum, with darker colors indicating higher abundance. Inner rings show sample-specific sorption rates of CIP (μg/day), with color intensity indicating increasing sorption capacity (light yellow = low, dark red = high). Phylum-level taxonomy is color-coded as indicated in the legend.