Ecological insights into low-level antibiotics interfered biofilms of Synechococcus elongatus

Abstract

The interactions between antibiotics and microorganisms have attracted considerable interests. As reported, low-level concentrations of antibiotics have been detected in various environments. Although many studies reported the inducing effects of antibiotics on biofilms, few research discussed their impacts on the ecological functions of microorganisms with biogeochemical importance. In this study, we investigated the effects of low-levels of kanamycin on the biofilm formation of Synechococcus elongatus as a proof of principle study. The results indicated that the biofilms of S. elongatus would be promoted in the presence of kanamycin, and the related photosynthesis-mediated calcification, a fundamental bio-machinery contributing to the local and global carbon cycle, will probably be enhanced as well. We believe this study would offer new information to evaluate the environmental risks of antibiotics and inspire more investigations on the ecological impacts of emerging pollutants.

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Due to the widespread application of antimicrobial drugs, antibiotics have been detected in humans, livestock and the environment.¹ Different types of antibiotics, such as aminoglycosides, β-lactams, diaminopyrimidines, tetracyclines, quinolones, and macrolides, have been detected in various environmental niches, normally ranging from ng L⁻¹ to μg L⁻¹.^1–3 The ubiquitous appearance of antibiotics in rivers, lakes and soils, particularly the low-level concentrations of antibiotics, has become a critical issue for both human health and natural environments. Specifically, antibiotics would accelerate the evolution of antibiotic resistance genes (ARGs). The ARGs can horizontally transfer across species, including pathogens, thus jeopardizing the use of antibiotics on the microorganisms harboring ARGs.¹ In addition, the low-level concentrations of antibiotics can work as signal molecules and interfere with the genetic and physiological properties, as well as the biofilm formations of exposed microorganisms.^4–6

Biofilms are a matrix of microorganisms supported by the scaffolds constructed by extracellular polymeric substances.⁷ Because of their multi-cellular nature, biofilms are innately resistant to antimicrobial drugs.⁸ Besides their impact on medicine, biofilms also have participated in the plant–bacteria interactions^9,10 and wastewater treatment processes.¹¹ As a result, it has a broad relevance to understanding how ubiquitous low-levels of antibiotics influence biofilms.

Previous studies reported the effects of antibiotics on biofilms as well as how microorganisms in biofilms fight against the antibiotics. Most studies used health-related or model microorganisms, such as Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, and Escherichia coli.^5,9,12–14 However, these organisms usually cannot represent the microorganisms with ecological importance. Cyanobacterium is one of the core participants in the biogeochemical cycles on Earth.^15–17 In particular, the photosynthesis-mediated calcification (PMC) regulates the biogenesis of carbonates, and it is one of the most fundamental processes in the local and global carbon cycle through history. About 41.9% of the total carbon was attributed to carbonate, and a quite large proportion of carbonate is of biogenic origin via PMC. The PMC relies largely on cyanobacterial biofilms.¹⁷ Given the existence of antibiotics in environments, cyanobacteria may also be exposed to low-level antibiotics. Therefore, it is important to know whether the interfered biofilm formation affects the biofilm-related ecological roles.

In this study, we performed a proof of principle study and investigated the effects of an aminoglycoside antibiotic, kanamycin, on the biofilm formation of the model cyanobacterium, Synechococcus elongatus PCC7942 (S. elongatus). Aminoglycoside antibiotics have been widely used in human therapy via a lethal mechanism, which elicits severe interference in the translation process of bacteria. Specifically, kanamycin is a typical aminoglycoside antibiotic and one of the most important medications. Besides medical applications, kanamycin is also used in molecular biology to isolate engineered strains. S. elongatus have been identified in various freshwater ecosystems, and the physiological, genetic and metabolic properties have been well studied. The PMC of S. elongatus was intensively investigated in previous studies. After evaluation of the effects of kanamycin on the biofilm formation, the subsequent impacts on the PMC were discussed via a combined transcriptional analysis. We believe that our observations can promote the understanding of the ecological impacts of antibiotics and inspire more research on the ecological influences of emerging pollutants.

PMC is one of the two main working mechanisms via which cyanobacteria contribute to the carbon cycle.^15,18,19 In the PMC, biofilms surrounding cyanobacteria together with the cell surfaces provide negative charge and various functional groups for calcium to bind and offer particular microenvironments for calcium to be precipitated. Thus, to evaluate the influences of low-level antibiotics on the biofilm-related ecological functions of cyanobacteria, we first examined whether low-level antibiotics would change the biofilm formations of S. elongatus.

For cells inoculated from early exponential phases, the formation of biofilms without antibiotics was quantified as 0.776 ± 0.342 and was regarded as the negative control. The biofilm formation increased to 2.589 ± 0.362 (334%) under 0.05 μg mL⁻¹ kanamycin (Fig. 1A). In the presence of 0.1 μg mL⁻¹ kanamycin, the biomass in biofilms increased from 0.776 ± 0.342 to 1.931 ± 0.138 (249%). Both increases were verified as significant by statistical analysis, indicating that low-level kanamycin would promote the formation of cyanobacterial biofilms. When the concentration of kanamycin reached up to 0.15 μg mL⁻¹, the increased biofilms cannot be verified by statistical analysis, which might be attributed to the inhibitory effects of kanamycin at this concentration. These results were supported by previous studies that concluded the universal inducing effects of low concentrations of antibiotics on biofilms.^4,5 We further identified the similar inducing effects of low-level antibiotics on cyanobacterial biofilms inoculated from the stationary phase. However, the biomass in biofilms increased only in the presence of 0.10 μg mL⁻¹ kanamycin compared with the control without kanamycin (Fig. 1B). This observation indicated that cyanobacteria from a different growth phase responded differently to the low-level antibiotics due to the different physiological properties.

Fig. 1 Effects of low-level antibiotics on the formation of biofilms of S. elongatus. S. elongatus cells were cultivated to either early exponential or stationary phase and then inoculated to 12-well plates for 6 days before analyzing the biomass in biofilms via measuring the amount of chlorophyll a by spectrophotometric method (detailed methods were listed in ESI†). The biomass was standardized via 1.0 OD₇₃₀ of S. elongatus cells. Panel (A) represents the biofilm of S. elongatus inoculated from early exponential phase, whereas panel (B) shows those inoculated from stationary phase. All experiments were conducted at least in triplicate and the error bars denote +1 SD from the means of independent experiments. The differences between data were evaluated using the Student's t-tests with P < 0.05 (*) as a significant difference.

Then, we examined whether the interfered biofilms along with the physiology affects the ecological functions of cyanobacteria. To this end, the transcriptional levels of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase (CA) were measured. RuBisCO catalyzes the primary step of CO₂ fixation using CO₂ as substrates. As the atmospheric CO₂ level is not high enough to support the requirements of cell growth, carbon concentration mechanisms, which are regulated by CA, have evolved.²⁰ CA converts HCO₃⁻ to CO₂, thus largely increasing the CO₂ concentration inside cells. While HCO₃⁻ transports through the cell membrane, OH⁻ is pumped out. The pH in the microenvironments of biofilms would increase and cause precipitation of calcium and sequestration of CO₂ in the form of calcium carbonate.^17,20 As such, RuBisCO, CA, and biofilms are the three most important factors dominating the PMC (Fig. 2A).

Fig. 2 Overall scheme of the PMC (A) and the transcriptional analysis of rbcL (B and D) and icfA (C and E). For the transcriptional analysis, the RNA was extracted from the S. elongatus cultured with low-level kanamycin for 6 d and then was used as a template for the cDNA synthesis. RT-qPCR was performed in a 20 μL reaction mixture, and the 2^−ΔΔC_T method was used to quantify the expression levels of rbcL and icfA. Panel (B) and (C) represent the S. elongatus inoculated from exponential phase, whereas (D) and (E) show those inoculated from stationary phase. All experiments were conducted at least in triplicate and the error bars denote +1 SD from the means of independent experiments. The differences between data were evaluated using the Student's t-tests with P < 0.05 (*) as a significant difference and P < 0.001 (**) as a very significant difference.

In the present study, we employed the quantitative real-time polymerase chain reaction to monitor the expression levels of rbcL (encoding for the large unit of RuBisCO) and icfA (encoding for CA). The obtained data were analyzed via the 2^−ΔΔC_T method using the 16s rDNA sequence as the housekeeping gene.²¹ The results showed that the expression level of rbcL was significantly increased under 0.05 μg mL⁻¹ of kanamycin (3.16 ± 1.18 folds) (Fig. 2B), at which level the biofilm formation was also promoted (Fig. 1A). The increase of the icfA expression level (2.87 ± 0.16 folds) was also observed in the same condition (Fig. 2C). These results indicated that when the biofilm formation was promoted, the activities of RuBisCO and CA were induced as well, which might lead to an up-regulated calcification activity.²² Under 0.10 μg mL⁻¹ of kanamycin, the expression levels of rbcL and icfA were increased marginally, although the biofilm formation was promoted. This suggested an interesting phenomenon that even the photosynthesis and CCM were not up-regulated and the calcification might be enhanced as the promoted biofilms provided better microenvironments and more binding sites for Ca²⁺ to be trapped.^16,17

Similar but distinct trends were observed for cells from stationary phases as well. Under 0.10 μg mL⁻¹ of kanamycin, the rbcL and icfA expression levels increased and the biofilms were also promoted (Fig. 2D and E), which may lead to the enhanced calcification. We also observed that under 0.05 μg mL⁻¹ of kanamycin, the expression levels of rbcL and icfA increased by 2.63 ± 0.66 folds and 1.30 ± 0.13 folds, respectively, although the biofilms varied slightly. This condition might also result in an increased level of calcification because photosynthesis and CCM were up-regulated while biofilms remained similar.

In conclusion, our study suggested that in the presence of 0.05 μg mL⁻¹ and 0.10 μg mL⁻¹ of kanamycin, PMC would be enhanced. These enhanced calcification potentials were caused by the combined effects of biofilm formation, photosynthesis, and CCM. Besides the importance as a carbon reservoir, the carbonate also contributes to the climates wherein the promoted precipitation of carbonate might increase the atmospheric CO₂ concentration.²³ Thus, the low-level antibiotics influence not only the formation of biofilms but also the biofilm-associated ecological functions of cyanobacteria. Nevertheless, these ecological effects interfered by low-level antibiotics may not be limited to cyanobacteria, but can also be applied to all microorganisms with biogeochemical functions. Overall, our study provides a new angle to think about antibiotics ecologically, and we believe that our work will inspire more investigations on microbial ecological roles of biofilms and the environmental risks of emerging pollutants.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

The authors thank Dr Guo-Chang Zhang, Dr Jing-Jing Liu and Dr Miao-Miao Liu for proofreading the main text. This work was supported by the National Natural Science Foundation of China (No. 21476130).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra15025j

‡ These authors contributed equally to this work.

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