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Response of microorganisms in biofilm to sulfadiazine and ciprofloxacin in drinking water distribution systems
Accepted Manuscript Response of microorganisms in biofilm to sulfadiazine and ciprofloxacin in drinking water distribution systems Haibo Wang, Chun Hu, Yi Shen, Baoyou Shi, Dan Zhao, Xueci Xing PII:
S0045-6535(18)32213-6
DOI:
https://doi.org/10.1016/j.chemosphere.2018.11.106
Reference:
CHEM 22590
To appear in:
ECSN
Received Date: 19 July 2018 Revised Date:
7 November 2018
Accepted Date: 15 November 2018
Please cite this article as: Wang, H., Hu, C., Shen, Y., Shi, B., Zhao, D., Xing, X., Response of microorganisms in biofilm to sulfadiazine and ciprofloxacin in drinking water distribution systems, Chemosphere (2018), doi: https://doi.org/10.1016/j.chemosphere.2018.11.106. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Graphical Abstract
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Response of microorganisms in biofilm to sulfadiazine and ciprofloxacin
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in drinking water distribution systems Haibo Wang,a
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Xueci Xingb
Chun Hu,a, b *
Yi Shen,c
Baoyou Shi,a, d
Dan Zhao,c **
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a
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Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China
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b
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Ministry of Education, Institute of Environmental Research at Greater Bay,
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Key Laboratory of Drinking Water Science and Technology, Research Center for
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Key Laboratory for Water Quality and Conservation of the Pearl River Delta,
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Guangzhou University, Guangzhou, 510006, China
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c
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and Technology, Suzhou, Jiangsu, 215009, China
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d
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School of Environmental Science and Engineering, Suzhou University of Science
University of Chinese Academy of Sciences, Beijing, 100049, China * Corresponding author Tel.: (+86)-10-62922155; Fax: (+86)-10-62843541
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E-mail address: [email protected] (Chun Hu), [email protected] (Dan Zhao)
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ABSTRACT
Effects of sulfadiazine and ciprofloxacin on microorganisms in biofilm of drinking
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water distribution systems (DWDSs) were studied. The results verified that the
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increases of 16S rRNA for total bacteria and bacterial genus Hyphomicrobium were
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related to the promotion of antibiotic resistance genes (ARGs) and class 1 integrons
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(int1) in DWDSs with sulfadiazine and ciprofloxacin. Moreover, the bacteria showed 1
ACCEPTED MANUSCRIPT higher enzymatic activities in DWDSs with sulfadiazine and ciprofloxacin, which
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resulted in more production of extracellular polymeric substances (EPS). The higher
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contents of EPS proteins and secondary structure β-sheet promoted bacterial
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aggregation and adsorption onto surface of pipelines to form biofilm. EPS can serve
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as a barrier for the microorganisms in biofilm. Therefore, the biofilm bacterial
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communities shifted and the 16S rRNA for total bacteria increased in DWDSs with
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antibiotics, which also drove the ARGs promotion. Furthermore, the two antibiotics
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exhibited stronger combined effects than that caused by sulfadiazine and
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ciprofloxacin alone.
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Keywords: sulfadiazine, ciprofloxacin, biofilm, antibiotic resistance genes,
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enzymatic activities, extracellular polymeric substances
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1. Introduction
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Supply of safe drinking water is very important for public health (Wang et al.,
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2013). Recently, antibiotic resistance bacteria (ARB) and antibiotic resistance genes
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(ARGs) have been extensively detected in drinking water and constitute a major
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public health issue (Bergeron et al., 2015; Binh et al., 2018). Disinfection removes the
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majority of microorganisms in source water, however, low level of microorganisms
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are still present in treated water before entering drinking water distribution systems
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(DWDSs) (Liu et al., 2016). Jia et al. (2015) have also found that bacterial community
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shift drives ARGs promotion during drinking water chlorination.
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ACCEPTED MANUSCRIPT When the drinking water goes into DWDSs, more than 90% of the total biomass
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exists as biofilm on the surface of pipelines, with only up to 5% of the biomass freely
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suspended in the bulk water (Liu et al., 2016; Lin et al., 2016). The detachment of
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biofilm in DWDSs will induce the deterioration of water quality at customers’ taps
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(Liu et al., 2017; Zhang et al., 2018). Biofilm formation in DWDSs is the result of
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bacterial attachment and multiplication of cells on the inner surface of pipelines (Xue
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et al., 2012; Fish et al., 2017). Biofilm formation in DWDSs is always affected by the
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water quality including natural organic matter (NOM), chlorine concentration,
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temperature and pH (Liu et al., 2016; Xue and Seo, 2013).
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Moreover, many antibiotics including sulfonamide and quinolone are detected in
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source water at level of µg L-1 or ng L-1 now (Johnson et al., 2015; Binh et al., 2018).
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Sulfadiazine and ciprofloxacin are the antibiotics of sulfonamide and quinolone
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usually found in source water, respectively (Gaffney et al., 2015; Binh et al., 2018).
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Trace level of these antibiotics, 1-4 ng L-1, has also been detected in drinking water
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due to ineffective removal through conventional processes (Ye and Weinberg, 2007;
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Gaffney et al., 2015; Jia et al., 2015). Antibiotics in the wastewater and drinking water
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can induce the change of microbial community and the promotion of ARGs (Tandukar
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et al., 2013; Jia et al., 2015; Harb et al., 2016; Wen et al., 2018). When trace level of
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antibiotics in the water go into DWDSs, antibiotics may also influence the biofilm
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composition and the changes of ARGs in biofilm. Jia et al. (2015) have found the
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linkages between bacterial community, ARGs and the concentration of antibiotics in
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drinking water, however, the effect mechanism of antibiotics on the change of
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bacterial community and ARGs in biofilm of DWDSs still remains unclear. In the presence of antibiotics, microbial stress responses may result in the changes
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in metabolism behaviors. Enzymes including dehydrogenase and protease are
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involved in the degradation of different organic matters (Han et al., 2016; Rai et al.,
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2017). Therefore, when the antibiotics are present in DWDSs, the antibiotics will
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affect enzymatic activities. Moreover, extracellular polymeric substances (EPS) are
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the bacterial metabolic products. Weathers et al. (2015) have found the increase of
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EPS production when bacteria were exposed to pollutants perfluoroalkyl acids with
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the concentration above 2 mg L-1. EPS can provide a protective barrier to the bacteria
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in biofilm, which increases the bacterial resistance to disinfectants. The mechanisms
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of EPS protection for bacteria include transport limitation of disinfectant through EPS
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matrix and sacrificial reaction of EPS with disinfectant (Xue et al., 2012). The
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composition and structure of EPS also play great roles on bacterial adsorption to form
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biofilm (Xue et al., 2012; Fish et al., 2016). The secondary structure of protein in EPS
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includes aggregated strands, β-sheet, random coil, α-helix, 3-turn helix and
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antiparallel β-sheet (Han et al., 2017). The effects of the secondary structure of
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protein in EPS on the biofilm formation in DWDSs are still unknown.
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Many studies have investigated the effects of antibiotics on bacterial community,
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and the effects of EPS on the aggregation ability of microorganisms and biofilm
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growth in wastewater and drinking water distribution systems (Jia et al., 2015; Hou et
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al., 2015; Liu et al., 2016; Jia et al., 2017). However, there are no reports about the
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relationship between the enzymatic activities, EPS production, the changes of 4
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bacterial community and ARGs, when the antibiotics are present in DWDSs.
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Therefore, the objectives of this study are (1) to investigate the 16S rRNA for total
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bacteria
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sulfadiazine/ciprofloxacin, and (2) to elucidate the effects mechanism of these
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antibiotics on biofilm by the analysis of enzymatic activity, EPS and ARGs.
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2. Materials and Methods
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2.1. Reagents and raw water
communities
in
biofilm
of
DWDSs
with
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bacterial
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and
Sulfadiazine and ciprofloxacin, high performance liquid chromatography grade,
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were purchased from Sigma-Aldrich Fluka (USA). Sodium hypochlorite solution,
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analytical grade, was obtained from Sinopharm Chemical Reagent Co., Ltd (China).
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The tested raw water was collected from a drinking water treatment plant in north
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of China, which was treated by coagulation using polyaluminium chloride,
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sedimentation, sand filtration, and biologically-activated carbon filtration (prior to
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entering the chlorine contact tanks). Every month, 250 L tested raw water was taken
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back to the laboratory from the drinking water treatment plant, and was stored at 4 °C
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before use during the experiments. Water quality parameters were measured according
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to standard methods (EPA of China, 2002), and the results were shown in Table S1.
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Differences of water quality were measured using analysis of variance (ANOVA) with
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a significance threshold of α=0.05. The sulfadiazine and ciprofloxacin were not
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detected
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Chromatography-Tandem Mass Spectrometer (UPLC-MS/MS, Quattro Premier XE,
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Waters, USA), therefore, the two antibiotics were added to the tested raw water
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in
the
tested
raw
water
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using
Ultra
Performance
Liquid
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during the experiments.
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2.2. Experiments set-up Ten cast iron coupons were immersed in covered 1.5 L glass fiber-reinforced plastic
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bottles to simulate the DWDSs. Before this study, twelve simulated DWDSs with the
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same format have been run at the same conditions for more than three years. During
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this period, the same tested raw water, which was chlorinated with 1 mg L-1 chlorine
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for 2 h, was added to the twelve simulated DWDSs, respectively. The water in each
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DWDS was displaced with chlorinated water at 48 h intervals and gently agitated by a
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magnetic rotor to mix the water, reflecting dead zones or worst case conditions in
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actual water distribution systems according to the reported methods (Liu et al., 2013;
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Wang et al., 2014).
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formed on surface of cast iron coupons. The cast iron coupons (80 mm×15 mm×5 mm)
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were purchased from Guangyuan Keyou Technology & Trade Co., Ltd (Beijing,
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China), and the surface area of each coupon is 12 cm2. The composition (wt%) was C
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3.25%, O 1.63%, Si 2.23%, P 0.08%, S 0.10%, Fe 90.48%, Cu 0.76%, Mn 0.72%, and
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Zn 0.75%.
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Sulfadiazine and ciprofloxacin at the level of ng L-1 have been found in tap water in
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some cities of China (Han et al., 2010; Jia et al., 2015). Moreover, sulfonamides and
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fluoroquinolones at the level of µg L-1 are always found in source water of China
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(Zhang et al., 2015). Therefore, four kinds of waters, including raw water, raw water
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with addition of 2 µg L-1 sulfadiazine, raw water with addition of 2 µg L-1 6
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ciprofloxacin, were used in this study. In the drinking water treatment plant from
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which the tested raw water was collected, 1 mg L-1 chlorine was used during the
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disinfection process. Therefore, 1 mg L-1 chlorine (NaClO solution) was also used in
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this study. After 1 L test water was chlorinated for 2 h with 1 mg L-1 chlorine, the four
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kinds of waters were poured into the DWDSs, respectively. The total chlorine
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concentration was measured using HANNA HI93711 spectrophotometer (Italy).
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The water in each DWDS was displaced with chlorinated water at 48 h intervals
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and gently agitated by a magnetic rotor to mix the water. Each experiment was done
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in triplicate. According to other studies and our previous studies (Wang et al., 2014;
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Wang et al., 2017), biofilm in simulated DWDSs can reach a relative stable state after
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8 months. Therefore, after 8 months, biofilm in each DWDS were sampled for only
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once, and then the 16S rRNA, ARGs, EPS, enzymatic activity and bacterial
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community in biofilm were analyzed immediately.
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2.3. Concentration analysis of sulfadiazine and ciprofloxacin
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When the sulfadiazine and ciprofloxacin were added to the raw water, the
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antibiotics in raw water, influents (chlorinated water) and effluents of the DWDSs
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were concentrated by solid phase extraction (SPE) method consisting of an HLB
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cartridge (200 mg (6 mL)-1) (Waters Oasis). The samples were taken every two weeks
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during this experiment for eight months. After taking the samples, the exact
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concentration of the antibiotics was immediately tested by Ultra Performance Liquid
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Chromatography-Tandem Mass Spectrometer (UPLC-MS/MS, Quattro Premier XE,
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Waters, USA). The detailed operation methods were listed in Text S1. The optimal
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conditions for the analysis of the two antibiotics were shown in Table S2.
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2.4. Sample collection and DNA extraction After removing the loose deposits by flushing, biofilm samples were scraped from
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10 cast iron coupons in DWDSs using sterile spatulas. The biofilm samples were
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brushed into 40 mL sterile phosphate-buffered saline (PBS, pH 7.0) and filtered
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through a 47 mm diameter, 0.2 µm polycarbonate membrane (Fish et al., 2017). The
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biofilm samples were taken in triplicate, therefore, 3 biofilm samples were brushed
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from 3 simulated DWDSs with the same kind of water to perform the 3 corresponding
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analysis.
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The propidium monoazide (PMA)-bound DNA cannot be amplified in the ensuing
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polymerase chain reactions (PCR). This characteristic is often applied to quantify the
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DNA of live bacteria and characterize the changes in viable bacterial communities
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(Gensberger et al., 2014). The process of PMA treated samples was listed in Text S2.
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An optimum PMA concentration of 40 µM was determined after testing a range of
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concentrations to maximize removal of DNA from 70%-isopropanol-killed
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Escherichia coli cells, while minimizing DNA removal from live cells (Fig. S1). This
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optimum PMA concentration of 40 µM was validated on untreated and
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isopropanol-treated (70%) (30 min incubation) cells from biofilm of DWDSs with
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raw water (Fig. S2). After PMA treatment, samples were subjected to DNA extraction
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with FastDNA SPIN Kit (MP Biomedicals, Solon, OH, USA) following the
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manufacturer’s instructions. To determine the method recovery efficiency, E. coli was
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2017). The recovery efficiency varied from 19.1% to 40.5% depending on the
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concentration of samples. Concentrations of DNA were measured with a Nanodrop
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spectrophotometer (ND-1000, NanoDrop, USA).
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2.5. Quantitative PCR and sequencing analysis
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The quantitative PCR (qPCR) experiments were carried out with ABI 7300 Fast
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Real-Time PCR System (Applied Biosystems, Singapore) using premix EX Taq or
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SYBR premix EX Taq (TaKaRa, Japan) in 20 µL reaction volume. The 16S rRNA for
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total bacteria (Wang et al., 2014), the efflux pump which encoding the membrane
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fusion mexA (Tandukar et al., 2013), class 1 integrons (int1) which strictly correlated
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to co-selection mechanism (Cesare et al., 2016), the sulfadiazine resistance genes
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(sul1, sul2, sul3) (Chen et al., 2015) and the ciprofloxacin resistance genes (qnrB and
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qnrS) (Wang et al., 2017) were quantified by qPCR. Primer sequences are presented
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in Table S3. Standard curves were generated with serial ten-fold dilution (109 to 102
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copies µL-1) of the plasmids. The analysis procedures and amplification efficiency
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were described in Text S3. The amplification efficiency values for quantification were
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from 96.5% to 98.8%. The limit of quantification (LOQ) for all qPCR assays ranged
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from 1 to 10 gene copies per reaction and was implemented as appropriate for each
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specific run.
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To determine the diversity and composition of biofilm bacterial communities in
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different DWDSs, PCR amplications were conducted in triplicate with 341f
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(5’-TTACCGCGGCTGCTGGCAC-3’) and 806r (5’-GGACTACNNGGGTATCTAAT 9
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2011). The PCR process was described in Text S4. After purification of PCR products
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and addition of index codes, sequencing libraries were generated. The library was
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sequenced on an Illumina HiSeq platform and 250 bp paired-end reads were generated
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at Novogene (Beijing, China). After sequencing, the sequences were analyzed,
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including operational taxonomic unit (OTU) clustering and taxonomic classification.
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The analysis process was listed in Text S5. Correlations analysis between the bacterial
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communities, 16S rRNA and ARGs was performed using SPSS (Inc., in Chicago,
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Illinois) version 19.0 for windows.
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2.6. Enzyme assays
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The biofilm samples were brushed into 40 mL sterile PBS (pH 7.0), and filtered
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through a 47 mm diameter, 0.2 µm polycarbonate membrane. Then, the enzymes in
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biofilm samples were determined. Dehydrogenase activity was assessed by a modified
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method in which 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride
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(INT) was used as a terminal hydrogen acceptor (Han et al., 2016). 2 mL of Tris-HCl
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buffer, 2 mL of Milli-Q water and 2 mL of 500 mg L-1 INT solution were added to the
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filters, sequentially. INT was changed to a red insoluble triphenyl formazan (TF)
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crystal by dehydrogenase. After incubation at 37 oC for 30 min, 0.1 mL H2SO4 (98%)
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was added into the tube to stop the reaction. Then, 5 mL acetone (99.5%) was added
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to the solution, and the supernatant was obtained by centrifugation (4000 rpm min-1,
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90 oC, 5 min) and measured at 485 nm using a spectrophotometer (Shimadzu
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UV-1800, Japan). The protease activity was assayed by a modified method using
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L-1 casein were added to the filters. The solutions were incubated at 40 oC for 10 min,
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and then added 2 mL of 0.4 M trichloroacetic acid. The content was centrifuged and
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the supernatant was analyzed using a spectrophotometer (Shimadzu UV-1800, Japan).
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The dehydrogenase and protease activity is expressed as µg of TF and tyrosine
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liberated h-1 cm-2 biofilm, respectively.
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2.7. EPS extraction and analysis
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A heat extraction method was modified to extract different EPS in biofilm of
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different DWDSs (Zhang et al., 2016). The biofilm samples were brushed into 50 mL
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centrifuge tubes with 40 mL sterile PBS (pH 7.0). The tubes were sonicated at 20
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KHz and 40 W for 30 s, followed by heating in water bath at 70 oC for 1 h, and then
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centrifuged at 8000 g for 20 min at 4 oC. The supernatant in the tubes were filtered
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through 0.45 µm polycarbonate filter to collect EPS. The proteins in EPS were
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determined with the Lowry procedure using bovine serum albumin (BSA) (Sigma) as
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standard. Fourier transform infrared spectroscopy (FTIR) (Bruker, Tensor 27) was
236
used to determine EPS structures and the distributions of the functional groups. The
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amide I region in the EPS was further analyzed to extract information regarding
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protein secondary structures using Peakfit software (version 4.12, Seasolve Software
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Inc.).
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3. Results and Discussion
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3.1. Changes of sulfadiazine and ciprofloxacin concentration
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Four kinds of DWDSs with raw water, sulfadiazine, ciprofloxacin, and 11
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that the actual concentration of sulfadiazine and ciprofloxacin was (1.94±0.16) µg L-1
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and (2.02±0.15) µg L-1, when they were added to the raw water, respectively (Table
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S4). However, when the waters were chlorinated for 2 h by 1 mg L-1 chlorine, the
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concentration of sulfadiazine became (10.7±1.08) ng L-1, and ciprofloxacin
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disappeared. Moreover, both of sulfadiazine and ciprofloxacin were not detected in
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effluents of DWDSs. Similarly, when sulfadiazine and ciprofloxacin were added to
250
the raw water simultaneously, the actual concentration was (1.03±0.08) µg L-1 and
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(0.99±0.07) µg L-1, respectively. After chlorination, only sulfadiazine was detected,
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and its concentration became (5.18±0.43) ng L-1 in the influents. Sulfadiazine and
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ciprofloxacin were also not detected in effluents of DWDSs.
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The results indicated that sulfadiazine and ciprofloxacin could react with chlorine,
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which induced the decrease of sulfadiazine concentration and the disappearance of
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ciprofloxacin in the influents of DWDSs. Moreover, the antibiotics and their
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chlorination byproducts may go through biotransformation by the bacteria in the
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DWDSs, resulting in the disappearance of these antibiotics in effluents of DWDSs
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(Wang et al., 2017). However, the total chlorine concentrations were 0.50 mg L-1 and
260
0.05 mg L-1 in the influents and effluents of the four kinds of DWDSs, respectively.
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They did not take great changes when the antibiotic added to the raw water due to the
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low concentration of these antibiotics (p>0.05).
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3.2. The total bacteria and ARGs in biofilm of different DWDSs
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The antibiotics in DWDSs would affect the biofilm formation and the ARGs 12
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sul3), the ciprofloxacin resistance genes (qnrB and qnrS), efflux pump gene mexA,
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and class 1 integrons (int1) in biofilm of different DWDSs were quantified by qPCR
268
(Fig. 1). The average gene copy numbers of 16S rRNA for total bacteria were
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(9.52±0.31), (10.9±0.33), (10.2±0.29), and (11.5±0.32) log (gene copies cm-2) in
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biofilm
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sulfadiazine-ciprofloxacin, respectively (Fig. 1a). The results indicated that trace level
272
of sulfadiazine and ciprofloxacin induced the increase of total bacteria in biofilms of
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DWDSs, and the combined effects of sulfadiazine and ciprofloxacin was higher than
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that resulted from sulfadiazine or ciprofloxacin alone.
DWDSs
with
raw
water,
sulfadiazine,
ciprofloxacin
and
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The average gene copy numbers of sul1 were (3.99±0.06), (5.40±0.12), (4.38±0.12),
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and (4.73±0.13) log (gene copies cm-2) in biofilm of DWDSs with raw water,
277
sulfadiazine, ciprofloxacin and sulfadiazine-ciprofloxacin, respectively (Fig. 1b). The
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gene copy number of sul1 was higher than that of sul2 and sul3, and the gene copy
279
numbers of sul1, sul2 and sul3 correlated very well (r>0.98, p<0.05) (Table S5). In
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addition, the average gene copy numbers of qnrS were (1.55±0.03), (3.34±0.05),
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(3.88±0.04), and (3.64±0.05) log (gene copies cm-2) in biofilm of the four kinds of
282
DWDSs. The gene copy number of qnrB showed the same changes with qnrS. The
283
results indicated that the higher concentration of sulfadiazine and ciprofloxacin
284
induced the higher increase of sulfadiazine and ciprofloxacin resistance genes,
285
respectively, which suggested that the concentrations of these antibiotics were the
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main determinants of sul1, sul2, sul3, qnrB and qnrS promotion.
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(5.41±0.15) and (5.61±0.18) log (gene copies cm-2) in biofilm of DWDSs with raw
289
water, sulfadiazine, ciprofloxacin and sulfadiazine-ciprofloxacin, respectively.
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Ciprofloxacin induced higher increase of mexA than that caused by sulfadiazine. The
291
multiple mex efflux genes can confer bacterial resistance to nearly all the antibiotics
292
by pumping out the antibiotics (Jia et al., 2015; Tandukar et al., 2013). Therefore, the
293
combined effect of sulfadiazine and ciprofloxacin resulted in the highest promotion of
294
mexA. In addition, the gene copy number of int1 were (4.31±0.11), (4.61±0.15),
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(4.43±0.13) and (5.17±0.16) log (gene copies cm-2) in biofilm of the four kinds of
296
DWDSs. An integron such as int1 is a typical gene capture and dissemination system,
297
which is nearly ubiquitous in various DNA materials (Chen et al., 2015; Wu et al.,
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2016). Bacteria with int1 had a selective advantage compared to other bacteria, and
299
int1 was strictly correlated to co-selection mechanisms (Cesare et al., 2016).
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Therefore, sulfadiazine and ciprofloxacin induced the increase of int1 obviously, and
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the combined effect of sulfadiazine and ciprofloxacin was stronger than that induced
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by sulfadiazine or ciprofloxacin alone.
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The gene copy numbers of 16S rRNA and int1 correlated very well (r>0.95, p<0.05)
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(Table S5). Integrons such as int1 play a great role in the horizontal gene transfer of
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ARGs between microbes (Gaze et al., 2011). Some studies also indicated that the shift
306
of bacterial community due to the antibiotics could promote the bacterial resistance
307
(Jia et al., 2015; Davids et al., 2017). Therefore, the promotion of the ARGs in biofilm
308
of different DWDSs, which was induced by the addition of sulfadiazine and 14
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ciprofloxacin, may be related with the increase of the total bacteria and the bacterial
310
community shift in biofilm.
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3.3. Bacterial community shift in biofilm of different DWDSs Based on the sequencing analysis of 16S rRNA genes, Proteobacteria phylum
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dominated in the biofilm bacterial communities of the four kinds of DWDSs, and its
314
relative abundance in all the biofilm bacterial communities was more than 89.7% (Fig.
315
S3a). At class level, Alphaproteobacteria was the main class in all the biofilm
316
bacterial communities (Fig. S3b). Furthermore, the dominant bacteria were compared
317
at genus level for different samples (Fig. 2). Hyphomicrobium, Sphingopyxis,
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Sphingomonas, Rhodobacter and Parvularcula were the predominant bacterial genera
319
in biofilm bacterial communities of DWDSs with raw water. The relative abundance
320
of Sphingopyxis and Parvularcula decreased in biofilm bacterial communities of
321
DWDSs with sulfadiazine, ciprofloxacin, and sulfadiazine-ciprofloxacin, which may
322
be due to the antibiotic activity of these antibiotics. The relative abundance of
323
Rhodobacter did not take great changes in DWDSs with the addition of sulfadiazine
324
and ciprofloxacin. However, compared with the DWDSs with raw water, the relative
325
abundance of Hyphomicrobium increased from 40.3% to 46.7%, 43.3% and 49.0% in
326
DWDSs with sulfadiazine, ciprofloxacin, and sulfadiazine-ciprofloxacin, respectively.
327
Moreover, the relative abundance of Bosea, Bdellovibrio and Sphingomonas also
328
increased in biofilm bacterial communities of DWDSs with these antibiotics.
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Compared with DWDSs with raw water, the 16S rRNA for total bacteria and the
330
relative abundance of Hyphomicrobium, Bosea, Bdellovibrio and Sphingomonas 15
ACCEPTED MANUSCRIPT increased in biofilm of DWDSs with antibiotics. The results suggested that the growth
332
of these bacterial genera was contributed to the increase of total bacteria in biofilm.
333
Moreover, the relative abundance of the main bacterial genus Hyphomicrobium
334
correlated very well with the gene copy numbers of 16S rRNA and int1 (r>0.95,
335
p<0.05) (Table S5). Therefore, the growth of bacterial genus Hyphomicrobium and the
336
increase of total bacteria were related with the antibiotic resistance genes in DWDSs
337
with sulfadiazine and ciprofloxacin. Moreover, the bacterial communities shift and the
338
total bacteria increase in biofilm induced the ARGs promotion, which was also
339
consistent with other studies indicating that bacterial community shift drove antibiotic
340
resistance promotion during drinking water chlorination (Jia et al., 2015).
341
3.4. Microbial metabolism behaviors
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The protease activity was (22.1±1.95), (29.3±2.05), (40.4±3.07) and (55.8±4.01) µg
343
tyrosine liberated h-1 cm-2 biofilm in DWDSs with raw water, sulfadiazine,
344
ciprofloxacin, and sulfadiazine-ciprofloxacin, respectively (Fig. 3). Moreover, the
345
dehydrogenase activity was (18.5±1.56), (36.0±3.08), (33.0±2.87) and (55.5±3.92) µg
346
TF liberated h-1 cm-2 biofilm in the four kinds of DWDSs, respectively. The results
347
indicated that trace level of sulfadiazine and ciprofloxacin increased the activity of
348
protease and dehydrogenase, and the combined effect of sulfadiazine and
349
ciprofloxacin induced the highest increase, compared with sulfadiazine or
350
ciprofloxacin alone.
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351
The degradation of complex polymeric organic matter by bacteria is related with
352
enzymatic activities (Lautenschlager et al., 2014; Han et al., 2016). Specific 16
ACCEPTED MANUSCRIPT hydrolytic enzymes (protease and dehydrogenase) are important for the growth of
354
microorganisms, when they were exposed to different organic matter including
355
antibiotics (Han et al., 2016; Rai et al., 2017). When antibiotics and their chlorination
356
products were present in DWDSs, they may affect the bacterial growth because of
357
their antibacterial activity. In this condition, metabolism behaviors of bacteria may
358
change due to the microbial stress responses. Therefore, protease and dehydrogenase
359
activities were affected sharply by the presence of antibiotics in our study.
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The dehydrogenase activity correlated very well with bacterial genus Bosea (r>0.97,
361
p<0.05) (Table S5). The dehydrogenase activity also showed the same tendency with
362
the main bacterial genus Hyphomicrobium. Moreover, the dehydrogenase activity
363
correlated very well with the gene copy numbers of 16S rRAN and int1 (r>0.95,
364
p<0.05). The protease activity also correlated very well with the gene copy number of
365
mexA (r>0.97, p<0.05) (Table S5). Therefore, the enzymatic activity of biofilm
366
bacterial communities might induce the increase of total bacteria in biofilm of
367
DWDSs with sulfadiazine and ciprofloxacin, and the increase of total bacteria also
368
induced the promotion of ARGs in biofilm. Moreover, EPS are involved in
369
extracellular electron transfer, and the presence of EPS can affect the enzymatic
370
activity (Han et al., 2017). Meanwhile, microbial metabolism behaviors may also
371
influence the EPS production. Therefore, the characterization of EPS in biofilm of
372
different DWDSs was also analyzed.
373
3.5. Roles of EPS on the biofilm formation
374
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Proteins were the main compositions of EPS, and the concentration of EPS proteins 17
ACCEPTED MANUSCRIPT were (194.7±7.36), (210.4±7.58), (198.4±7.14) and (244.0±7.18) µg cm-2 in biofilm
376
of DWDSs with raw water, sulfadiazine, ciprofloxacin, sulfadiazine-ciprofloxacin,
377
respectively (Fig. 4). The results indicated that the combined effects of sulfadiazine
378
and ciprofloxacin induced the highest increase of EPS proteins, followed by
379
sulfadiazine and ciprofloxacin alone.
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Several regions on the FTIR spectra provided information regarding major
381
functional moieties of EPS (Hessler et al., 2012). FTIR spectra showed that nine
382
peaks were present in EPS (Fig. 5a). The peaks at 1049 and 1076 cm-1 were related to
383
C-O stretching of polysaccharides (Adeleye and Keller, 2016). The peak at 1248 cm-1
384
was related to P=O stretching of phospholipids or nucleic acids (Adeleye and Keller,
385
2016). The bands at 1410 cm-1 and 1452 cm-1 corresponded to C–H bending and C–N
386
stretching vibrations of proteins, respectively, which may arise from the amide II (You
387
et al., 2015). The peak bands (1600-1500 cm-1) were due to N-H bending and C-N
388
stretching vibrations in proteins (amide II band), which was very weak in EPS
389
(Adeleye and Keller, 2016). The amide I band (1700-1600 cm-1) was attributable to
390
the C=C and C=O stretching in proteins (Hessler et al., 2012; Jia et al., 2017). The
391
peaks at 2897 and 2972 cm-1 were assigned to C-H antisym and sym stretching in
392
lipids (Adeleye and Keller, 2016). Moreover, for elucidating the changes of the
393
secondary structure of proteins, the curve fitting of original infrared spectra for amide
394
I region were performed (Fig. 5b and Fig. S4). The protein secondary structures
395
included aggregated strands, β-sheet, random coil, α-helix, 3-turn helix and
396
antiparallel β-sheet (Han et al., 2017). The relative contents of these secondary
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398
raw water, the relative contents of β-sheet in EPS from biofilm increased from 20.3%
399
to 25.6%, 22.1% and 27.6% in DWDSs with sulfadiazine, ciprofloxacin and
400
sulfadiazine-ciprofloxacin, respectively. The changes of the relative contents of
401
β-sheet showed the same tendency with the proteins concentration in EPS. The
402
combined effects of sulfadiazine and ciprofloxacin induced the highest increase of
403
β-sheet, followed by sulfadiazine and ciprofloxacin alone.
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As we all known, EPS played great roles on the bacterial irreversible adherence
405
onto surface to form biofilm (Xue et al., 2012; Fish et al., 2016). In our study,
406
sulfadiazine and ciprofloxacin induced more production of EPS with higher contents
407
of proteins and β-sheet. The relative abundance of Hyphomicrobium and Bdellovibrio
408
correlated very well with β-sheet (r>0.95, p<0.05) (Table S5). Moreover, the gene
409
copy numbers of 16S rRNA and int1 correlated very well with the EPS proteins and
410
β-sheet, respectively (r>0.95, p<0.05) (Table S5). The gene copy numbers of 16S
411
rRNA and int1 also showed good relationship with the enzymatic activities (r>0.95,
412
p<0.05) (Table S5). Therefore, the total bacteria exhibited the highest enzymatic
413
activities in DWDSs with sulfadiazine-ciprofloxacin and induced the highest EPS
414
production. The higher contents of proteins increased the hydrophobicity of cell
415
surfaces (Jia et al., 2017). Due to the twisted and pleated sheet structure of β-sheet,
416
large amounts of inner hydrophobic groups of amino acids were more easily to be
417
exposed and express the hydrophobic property of the bacteria (Hou et al., 2015). The
418
increased hydrophobicity of bacterial surfaces due to the higher contents of proteins
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420
pipelines to form biofilm. The results were consistent with other studies which
421
indicated that the secondary structure of protein in EPS such as β-sheet played
422
important roles in bioflocculation and adhesion (Yin et al., 2015; Jia et al., 2017).
423
Moreover, EPS can serve as a barrier to mitigate the disinfectant and antibiotics
424
intrusion into the bacteria (Han et al., 2017). Therefore, the bacterial communities
425
changed and the relative abundance of Hyphomicrobium enhanced, meanwhile, the
426
gene copy number of 16S rRNA for total bacteria also increased in biofilm of DWDSs
427
with sulfadiazine and ciprofloxacin, which induced the ARGs promotion. The
428
combined effect of sulfadiazine and ciprofloxacin was stronger than that resulted from
429
sulfadiazine and ciprofloxacin alone.
430
4. Conclusions
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Compared with DWDSs with raw water, the gene copy numbers of 16S rRNA for
432
total bacteria and the ARGs and class 1 integrons (int1) increased in DWDSs with
433
sulfadiazine and ciprofloxacin. The relative abundance of bacterial genus
434
Hyphomicrobium also enhanced. The increase of total bacteria was related to the
435
ARGs promotion. Moreover, the bacteria exhibited higher enzymatic activities and
436
produced more EPS in DWDSs with sulfadiazine and ciprofloxacin. The higher
437
contents of EPS proteins and secondary structure β-sheet promoted bacterial
438
adsorption onto surface of pipelines to form biofilm. Therefore, the total bacteria
439
increased in DWDSs with antibiotics, which also drove ARGs promotion.
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Acknowledgments This work was funded by the National Natural Science Foundation of China (Nos. 51878654,
444
(QYZDY-SSW-DQC004) and the Federal Department of Chinese Water Control and
445
Treatment (Nos. 2017ZX07108, 2017ZX07501002).
446
Declaration of interest
449
project
of
None
Chinese
Academy
of
Sciences
SC
448
the
Appendix A. Supplementary data
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51838005),
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Supplementary material related to this article can be found in the online version.
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Reference
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Table Captions
3
Table 1. The relative content of each secondary structure in amide I region of EPS
4
proteins from biofilm bacterial communities in different drinking water distribution
5
systems (%).
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6
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1
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Table 1. The relative content of each secondary structure in amide I region of EPS
13
proteins from biofilm bacterial communities in different drinking water distribution
14
systems (%). protein secondary
SC
12
Raw
Two
Sulfadiazine Ciprofloxacin water
aggregated strands
7.28
9.64
8.16
5.72
β-sheet
20.3
25.6
22.1
27.6
random coil
26.7
21.5
16.6
23.6
α-helix
29.1
26.9
35.5
27.7
3-turn helix
10.8
12.4
12.8
9.13
5.74
4.03
4.87
6.22
17 18 19 20
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antibiotics
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Figure Captions
3
Fig. 1. The gene copy numbers of 16S rRNA for total bacteria (a) and the antibiotic
4
resistance genes (b) in biofilm of different drinking water distribution systems. Error
5
bars represent the standard deviation from the average of three replications.
6
Fig. 2. The relative abundance of main bacterial genera in biofilm of different
7
drinking water distribution systems. Other bacterial genera, relative abundance of
8
which was lower than 1% in all the samples, were not shown.
9
Fig. 3. The enzymatic activities of protease and dehydrogenase in biofilm of different
10
drinking water distribution systems. Error bars represent the standard deviation from
11
the average of three replications.
12
Fig. 4. The proteins concentration in EPS from biofilm bacterial community of
13
different drinking water distribution systems. Error bars represent the standard
14
deviation from the average of three replications.
15
Fig. 5. The FTIR spectra of EPS from biofilm bacterial community in different
16
drinking water distribution systems (a), and the secondary structures and curve-fitted
17
amide I region (1700-1600 cm-1) of EPS in biofilm of DWDSs with raw water (b).
SC
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(a)
10
6 4 2 0
e cin iotics water diazin floxa antib Raw Sulfa Cipro Two
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5 4
2 1
mexA int1
sul1
sul2
sul3 qnrB qnrS
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Raw water Sulfadiazine Ciprofloxacin Two antibiotics
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(b)
6
-2
Log (gene copies cm biofilm)
20
21
16S rRNA
SC
log (gene copies cm-2 biofilm)
19
22
Fig. 1. The gene copy numbers of 16S rRNA for total bacteria (a) and the antibiotic
23
resistance genes (b) in biofilm of different drinking water distribution systems. Error
24
bars represent the standard deviation from the average of three replications.
25
2
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31
60
Rhizobacter Parvularcula Rhodobacter Sphingomonas Bdellovibrio Bosea Methylobacterium Sphingopyxis Hyphomicrobium
50
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70
40 30 20 10 0
32
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Fig. 2. The relative abundance of main bacterial genera in biofilm of different
34
drinking water distribution systems. Other bacterial genera, relative abundance of
35
which was lower than 1% in all the samples, were not shown.
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Enzymatic activity (µg h cm biofilm)
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Fig. 3. The enzymatic activities of protease and dehydrogenase in biofilm of different
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drinking water distribution systems. Error bars represent the standard deviation from
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the average of three replications.
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Fig. 4. The proteins concentration in EPS from biofilm bacterial community of
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different drinking water distribution systems. Error bars represent the standard
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deviation from the average of three replications.
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Fig. 5. The FTIR spectra of EPS from biofilm bacterial community in different
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drinking water distribution systems (a), and the secondary structures and curve-fitted
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amide I region (1700-1600 cm-1) of EPS in biofilm of DWDSs with raw water (b).
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ACCEPTED MANUSCRIPT Highlights Response of biofilm to sulfadiazine and ciprofloxacin in DWDSs was studied. Bacterial community shift and total bacteria increase related to ARGs promotion.
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The bacteria exhibited higher enzymatic activity and produced more EPS. The higher contents of proteins and β-sheet in EPS promoted biofilm formation.
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Sulfadiazine and ciprofloxacin exhibited stronger combined effects on biofilm.
S0045-6535(18)32213-6
DOI:
https://doi.org/10.1016/j.chemosphere.2018.11.106
Reference:
CHEM 22590
To appear in:
ECSN
Received Date: 19 July 2018 Revised Date:
7 November 2018
Accepted Date: 15 November 2018
Please cite this article as: Wang, H., Hu, C., Shen, Y., Shi, B., Zhao, D., Xing, X., Response of microorganisms in biofilm to sulfadiazine and ciprofloxacin in drinking water distribution systems, Chemosphere (2018), doi: https://doi.org/10.1016/j.chemosphere.2018.11.106. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Graphical Abstract
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Response of microorganisms in biofilm to sulfadiazine and ciprofloxacin
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in drinking water distribution systems Haibo Wang,a
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Xueci Xingb
Chun Hu,a, b *
Yi Shen,c
Baoyou Shi,a, d
Dan Zhao,c **
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a
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Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China
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b
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Ministry of Education, Institute of Environmental Research at Greater Bay,
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Key Laboratory of Drinking Water Science and Technology, Research Center for
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Key Laboratory for Water Quality and Conservation of the Pearl River Delta,
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Guangzhou University, Guangzhou, 510006, China
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c
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and Technology, Suzhou, Jiangsu, 215009, China
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d
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School of Environmental Science and Engineering, Suzhou University of Science
University of Chinese Academy of Sciences, Beijing, 100049, China * Corresponding author Tel.: (+86)-10-62922155; Fax: (+86)-10-62843541
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E-mail address: [email protected] (Chun Hu), [email protected] (Dan Zhao)
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ABSTRACT
Effects of sulfadiazine and ciprofloxacin on microorganisms in biofilm of drinking
19
water distribution systems (DWDSs) were studied. The results verified that the
20
increases of 16S rRNA for total bacteria and bacterial genus Hyphomicrobium were
21
related to the promotion of antibiotic resistance genes (ARGs) and class 1 integrons
22
(int1) in DWDSs with sulfadiazine and ciprofloxacin. Moreover, the bacteria showed 1
ACCEPTED MANUSCRIPT higher enzymatic activities in DWDSs with sulfadiazine and ciprofloxacin, which
24
resulted in more production of extracellular polymeric substances (EPS). The higher
25
contents of EPS proteins and secondary structure β-sheet promoted bacterial
26
aggregation and adsorption onto surface of pipelines to form biofilm. EPS can serve
27
as a barrier for the microorganisms in biofilm. Therefore, the biofilm bacterial
28
communities shifted and the 16S rRNA for total bacteria increased in DWDSs with
29
antibiotics, which also drove the ARGs promotion. Furthermore, the two antibiotics
30
exhibited stronger combined effects than that caused by sulfadiazine and
31
ciprofloxacin alone.
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Keywords: sulfadiazine, ciprofloxacin, biofilm, antibiotic resistance genes,
34
enzymatic activities, extracellular polymeric substances
35 36
1. Introduction
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Supply of safe drinking water is very important for public health (Wang et al.,
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2013). Recently, antibiotic resistance bacteria (ARB) and antibiotic resistance genes
39
(ARGs) have been extensively detected in drinking water and constitute a major
40
public health issue (Bergeron et al., 2015; Binh et al., 2018). Disinfection removes the
41
majority of microorganisms in source water, however, low level of microorganisms
42
are still present in treated water before entering drinking water distribution systems
43
(DWDSs) (Liu et al., 2016). Jia et al. (2015) have also found that bacterial community
44
shift drives ARGs promotion during drinking water chlorination.
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ACCEPTED MANUSCRIPT When the drinking water goes into DWDSs, more than 90% of the total biomass
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exists as biofilm on the surface of pipelines, with only up to 5% of the biomass freely
47
suspended in the bulk water (Liu et al., 2016; Lin et al., 2016). The detachment of
48
biofilm in DWDSs will induce the deterioration of water quality at customers’ taps
49
(Liu et al., 2017; Zhang et al., 2018). Biofilm formation in DWDSs is the result of
50
bacterial attachment and multiplication of cells on the inner surface of pipelines (Xue
51
et al., 2012; Fish et al., 2017). Biofilm formation in DWDSs is always affected by the
52
water quality including natural organic matter (NOM), chlorine concentration,
53
temperature and pH (Liu et al., 2016; Xue and Seo, 2013).
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Moreover, many antibiotics including sulfonamide and quinolone are detected in
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source water at level of µg L-1 or ng L-1 now (Johnson et al., 2015; Binh et al., 2018).
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Sulfadiazine and ciprofloxacin are the antibiotics of sulfonamide and quinolone
57
usually found in source water, respectively (Gaffney et al., 2015; Binh et al., 2018).
58
Trace level of these antibiotics, 1-4 ng L-1, has also been detected in drinking water
59
due to ineffective removal through conventional processes (Ye and Weinberg, 2007;
60
Gaffney et al., 2015; Jia et al., 2015). Antibiotics in the wastewater and drinking water
61
can induce the change of microbial community and the promotion of ARGs (Tandukar
62
et al., 2013; Jia et al., 2015; Harb et al., 2016; Wen et al., 2018). When trace level of
63
antibiotics in the water go into DWDSs, antibiotics may also influence the biofilm
64
composition and the changes of ARGs in biofilm. Jia et al. (2015) have found the
65
linkages between bacterial community, ARGs and the concentration of antibiotics in
66
drinking water, however, the effect mechanism of antibiotics on the change of
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bacterial community and ARGs in biofilm of DWDSs still remains unclear. In the presence of antibiotics, microbial stress responses may result in the changes
69
in metabolism behaviors. Enzymes including dehydrogenase and protease are
70
involved in the degradation of different organic matters (Han et al., 2016; Rai et al.,
71
2017). Therefore, when the antibiotics are present in DWDSs, the antibiotics will
72
affect enzymatic activities. Moreover, extracellular polymeric substances (EPS) are
73
the bacterial metabolic products. Weathers et al. (2015) have found the increase of
74
EPS production when bacteria were exposed to pollutants perfluoroalkyl acids with
75
the concentration above 2 mg L-1. EPS can provide a protective barrier to the bacteria
76
in biofilm, which increases the bacterial resistance to disinfectants. The mechanisms
77
of EPS protection for bacteria include transport limitation of disinfectant through EPS
78
matrix and sacrificial reaction of EPS with disinfectant (Xue et al., 2012). The
79
composition and structure of EPS also play great roles on bacterial adsorption to form
80
biofilm (Xue et al., 2012; Fish et al., 2016). The secondary structure of protein in EPS
81
includes aggregated strands, β-sheet, random coil, α-helix, 3-turn helix and
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antiparallel β-sheet (Han et al., 2017). The effects of the secondary structure of
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protein in EPS on the biofilm formation in DWDSs are still unknown.
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Many studies have investigated the effects of antibiotics on bacterial community,
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and the effects of EPS on the aggregation ability of microorganisms and biofilm
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growth in wastewater and drinking water distribution systems (Jia et al., 2015; Hou et
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al., 2015; Liu et al., 2016; Jia et al., 2017). However, there are no reports about the
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relationship between the enzymatic activities, EPS production, the changes of 4
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bacterial community and ARGs, when the antibiotics are present in DWDSs.
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Therefore, the objectives of this study are (1) to investigate the 16S rRNA for total
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bacteria
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sulfadiazine/ciprofloxacin, and (2) to elucidate the effects mechanism of these
93
antibiotics on biofilm by the analysis of enzymatic activity, EPS and ARGs.
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2. Materials and Methods
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2.1. Reagents and raw water
communities
in
biofilm
of
DWDSs
with
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and
Sulfadiazine and ciprofloxacin, high performance liquid chromatography grade,
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were purchased from Sigma-Aldrich Fluka (USA). Sodium hypochlorite solution,
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analytical grade, was obtained from Sinopharm Chemical Reagent Co., Ltd (China).
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The tested raw water was collected from a drinking water treatment plant in north
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of China, which was treated by coagulation using polyaluminium chloride,
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sedimentation, sand filtration, and biologically-activated carbon filtration (prior to
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entering the chlorine contact tanks). Every month, 250 L tested raw water was taken
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back to the laboratory from the drinking water treatment plant, and was stored at 4 °C
104
before use during the experiments. Water quality parameters were measured according
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to standard methods (EPA of China, 2002), and the results were shown in Table S1.
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Differences of water quality were measured using analysis of variance (ANOVA) with
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a significance threshold of α=0.05. The sulfadiazine and ciprofloxacin were not
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detected
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Chromatography-Tandem Mass Spectrometer (UPLC-MS/MS, Quattro Premier XE,
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Waters, USA), therefore, the two antibiotics were added to the tested raw water
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in
the
tested
raw
water
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using
Ultra
Performance
Liquid
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during the experiments.
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2.2. Experiments set-up Ten cast iron coupons were immersed in covered 1.5 L glass fiber-reinforced plastic
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bottles to simulate the DWDSs. Before this study, twelve simulated DWDSs with the
115
same format have been run at the same conditions for more than three years. During
116
this period, the same tested raw water, which was chlorinated with 1 mg L-1 chlorine
117
for 2 h, was added to the twelve simulated DWDSs, respectively. The water in each
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DWDS was displaced with chlorinated water at 48 h intervals and gently agitated by a
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magnetic rotor to mix the water, reflecting dead zones or worst case conditions in
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actual water distribution systems according to the reported methods (Liu et al., 2013;
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Wang et al., 2014).
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formed on surface of cast iron coupons. The cast iron coupons (80 mm×15 mm×5 mm)
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were purchased from Guangyuan Keyou Technology & Trade Co., Ltd (Beijing,
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China), and the surface area of each coupon is 12 cm2. The composition (wt%) was C
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3.25%, O 1.63%, Si 2.23%, P 0.08%, S 0.10%, Fe 90.48%, Cu 0.76%, Mn 0.72%, and
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Zn 0.75%.
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Sulfadiazine and ciprofloxacin at the level of ng L-1 have been found in tap water in
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some cities of China (Han et al., 2010; Jia et al., 2015). Moreover, sulfonamides and
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fluoroquinolones at the level of µg L-1 are always found in source water of China
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(Zhang et al., 2015). Therefore, four kinds of waters, including raw water, raw water
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with addition of 2 µg L-1 sulfadiazine, raw water with addition of 2 µg L-1 6
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ciprofloxacin, were used in this study. In the drinking water treatment plant from
135
which the tested raw water was collected, 1 mg L-1 chlorine was used during the
136
disinfection process. Therefore, 1 mg L-1 chlorine (NaClO solution) was also used in
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this study. After 1 L test water was chlorinated for 2 h with 1 mg L-1 chlorine, the four
138
kinds of waters were poured into the DWDSs, respectively. The total chlorine
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concentration was measured using HANNA HI93711 spectrophotometer (Italy).
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The water in each DWDS was displaced with chlorinated water at 48 h intervals
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and gently agitated by a magnetic rotor to mix the water. Each experiment was done
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in triplicate. According to other studies and our previous studies (Wang et al., 2014;
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Wang et al., 2017), biofilm in simulated DWDSs can reach a relative stable state after
144
8 months. Therefore, after 8 months, biofilm in each DWDS were sampled for only
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once, and then the 16S rRNA, ARGs, EPS, enzymatic activity and bacterial
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community in biofilm were analyzed immediately.
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2.3. Concentration analysis of sulfadiazine and ciprofloxacin
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When the sulfadiazine and ciprofloxacin were added to the raw water, the
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antibiotics in raw water, influents (chlorinated water) and effluents of the DWDSs
150
were concentrated by solid phase extraction (SPE) method consisting of an HLB
151
cartridge (200 mg (6 mL)-1) (Waters Oasis). The samples were taken every two weeks
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during this experiment for eight months. After taking the samples, the exact
153
concentration of the antibiotics was immediately tested by Ultra Performance Liquid
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Chromatography-Tandem Mass Spectrometer (UPLC-MS/MS, Quattro Premier XE,
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Waters, USA). The detailed operation methods were listed in Text S1. The optimal
156
conditions for the analysis of the two antibiotics were shown in Table S2.
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2.4. Sample collection and DNA extraction After removing the loose deposits by flushing, biofilm samples were scraped from
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10 cast iron coupons in DWDSs using sterile spatulas. The biofilm samples were
160
brushed into 40 mL sterile phosphate-buffered saline (PBS, pH 7.0) and filtered
161
through a 47 mm diameter, 0.2 µm polycarbonate membrane (Fish et al., 2017). The
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biofilm samples were taken in triplicate, therefore, 3 biofilm samples were brushed
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from 3 simulated DWDSs with the same kind of water to perform the 3 corresponding
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analysis.
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The propidium monoazide (PMA)-bound DNA cannot be amplified in the ensuing
166
polymerase chain reactions (PCR). This characteristic is often applied to quantify the
167
DNA of live bacteria and characterize the changes in viable bacterial communities
168
(Gensberger et al., 2014). The process of PMA treated samples was listed in Text S2.
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An optimum PMA concentration of 40 µM was determined after testing a range of
170
concentrations to maximize removal of DNA from 70%-isopropanol-killed
171
Escherichia coli cells, while minimizing DNA removal from live cells (Fig. S1). This
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optimum PMA concentration of 40 µM was validated on untreated and
173
isopropanol-treated (70%) (30 min incubation) cells from biofilm of DWDSs with
174
raw water (Fig. S2). After PMA treatment, samples were subjected to DNA extraction
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with FastDNA SPIN Kit (MP Biomedicals, Solon, OH, USA) following the
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manufacturer’s instructions. To determine the method recovery efficiency, E. coli was
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2017). The recovery efficiency varied from 19.1% to 40.5% depending on the
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concentration of samples. Concentrations of DNA were measured with a Nanodrop
180
spectrophotometer (ND-1000, NanoDrop, USA).
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2.5. Quantitative PCR and sequencing analysis
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The quantitative PCR (qPCR) experiments were carried out with ABI 7300 Fast
183
Real-Time PCR System (Applied Biosystems, Singapore) using premix EX Taq or
184
SYBR premix EX Taq (TaKaRa, Japan) in 20 µL reaction volume. The 16S rRNA for
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total bacteria (Wang et al., 2014), the efflux pump which encoding the membrane
186
fusion mexA (Tandukar et al., 2013), class 1 integrons (int1) which strictly correlated
187
to co-selection mechanism (Cesare et al., 2016), the sulfadiazine resistance genes
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(sul1, sul2, sul3) (Chen et al., 2015) and the ciprofloxacin resistance genes (qnrB and
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qnrS) (Wang et al., 2017) were quantified by qPCR. Primer sequences are presented
190
in Table S3. Standard curves were generated with serial ten-fold dilution (109 to 102
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copies µL-1) of the plasmids. The analysis procedures and amplification efficiency
192
were described in Text S3. The amplification efficiency values for quantification were
193
from 96.5% to 98.8%. The limit of quantification (LOQ) for all qPCR assays ranged
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from 1 to 10 gene copies per reaction and was implemented as appropriate for each
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specific run.
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To determine the diversity and composition of biofilm bacterial communities in
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different DWDSs, PCR amplications were conducted in triplicate with 341f
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(5’-TTACCGCGGCTGCTGGCAC-3’) and 806r (5’-GGACTACNNGGGTATCTAAT 9
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200
2011). The PCR process was described in Text S4. After purification of PCR products
201
and addition of index codes, sequencing libraries were generated. The library was
202
sequenced on an Illumina HiSeq platform and 250 bp paired-end reads were generated
203
at Novogene (Beijing, China). After sequencing, the sequences were analyzed,
204
including operational taxonomic unit (OTU) clustering and taxonomic classification.
205
The analysis process was listed in Text S5. Correlations analysis between the bacterial
206
communities, 16S rRNA and ARGs was performed using SPSS (Inc., in Chicago,
207
Illinois) version 19.0 for windows.
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2.6. Enzyme assays
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The biofilm samples were brushed into 40 mL sterile PBS (pH 7.0), and filtered
210
through a 47 mm diameter, 0.2 µm polycarbonate membrane. Then, the enzymes in
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biofilm samples were determined. Dehydrogenase activity was assessed by a modified
212
method in which 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride
213
(INT) was used as a terminal hydrogen acceptor (Han et al., 2016). 2 mL of Tris-HCl
214
buffer, 2 mL of Milli-Q water and 2 mL of 500 mg L-1 INT solution were added to the
215
filters, sequentially. INT was changed to a red insoluble triphenyl formazan (TF)
216
crystal by dehydrogenase. After incubation at 37 oC for 30 min, 0.1 mL H2SO4 (98%)
217
was added into the tube to stop the reaction. Then, 5 mL acetone (99.5%) was added
218
to the solution, and the supernatant was obtained by centrifugation (4000 rpm min-1,
219
90 oC, 5 min) and measured at 485 nm using a spectrophotometer (Shimadzu
220
UV-1800, Japan). The protease activity was assayed by a modified method using
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222
L-1 casein were added to the filters. The solutions were incubated at 40 oC for 10 min,
223
and then added 2 mL of 0.4 M trichloroacetic acid. The content was centrifuged and
224
the supernatant was analyzed using a spectrophotometer (Shimadzu UV-1800, Japan).
225
The dehydrogenase and protease activity is expressed as µg of TF and tyrosine
226
liberated h-1 cm-2 biofilm, respectively.
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2.7. EPS extraction and analysis
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A heat extraction method was modified to extract different EPS in biofilm of
229
different DWDSs (Zhang et al., 2016). The biofilm samples were brushed into 50 mL
230
centrifuge tubes with 40 mL sterile PBS (pH 7.0). The tubes were sonicated at 20
231
KHz and 40 W for 30 s, followed by heating in water bath at 70 oC for 1 h, and then
232
centrifuged at 8000 g for 20 min at 4 oC. The supernatant in the tubes were filtered
233
through 0.45 µm polycarbonate filter to collect EPS. The proteins in EPS were
234
determined with the Lowry procedure using bovine serum albumin (BSA) (Sigma) as
235
standard. Fourier transform infrared spectroscopy (FTIR) (Bruker, Tensor 27) was
236
used to determine EPS structures and the distributions of the functional groups. The
237
amide I region in the EPS was further analyzed to extract information regarding
238
protein secondary structures using Peakfit software (version 4.12, Seasolve Software
239
Inc.).
240
3. Results and Discussion
241
3.1. Changes of sulfadiazine and ciprofloxacin concentration
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Four kinds of DWDSs with raw water, sulfadiazine, ciprofloxacin, and 11
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244
that the actual concentration of sulfadiazine and ciprofloxacin was (1.94±0.16) µg L-1
245
and (2.02±0.15) µg L-1, when they were added to the raw water, respectively (Table
246
S4). However, when the waters were chlorinated for 2 h by 1 mg L-1 chlorine, the
247
concentration of sulfadiazine became (10.7±1.08) ng L-1, and ciprofloxacin
248
disappeared. Moreover, both of sulfadiazine and ciprofloxacin were not detected in
249
effluents of DWDSs. Similarly, when sulfadiazine and ciprofloxacin were added to
250
the raw water simultaneously, the actual concentration was (1.03±0.08) µg L-1 and
251
(0.99±0.07) µg L-1, respectively. After chlorination, only sulfadiazine was detected,
252
and its concentration became (5.18±0.43) ng L-1 in the influents. Sulfadiazine and
253
ciprofloxacin were also not detected in effluents of DWDSs.
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The results indicated that sulfadiazine and ciprofloxacin could react with chlorine,
255
which induced the decrease of sulfadiazine concentration and the disappearance of
256
ciprofloxacin in the influents of DWDSs. Moreover, the antibiotics and their
257
chlorination byproducts may go through biotransformation by the bacteria in the
258
DWDSs, resulting in the disappearance of these antibiotics in effluents of DWDSs
259
(Wang et al., 2017). However, the total chlorine concentrations were 0.50 mg L-1 and
260
0.05 mg L-1 in the influents and effluents of the four kinds of DWDSs, respectively.
261
They did not take great changes when the antibiotic added to the raw water due to the
262
low concentration of these antibiotics (p>0.05).
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3.2. The total bacteria and ARGs in biofilm of different DWDSs
264
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The antibiotics in DWDSs would affect the biofilm formation and the ARGs 12
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266
sul3), the ciprofloxacin resistance genes (qnrB and qnrS), efflux pump gene mexA,
267
and class 1 integrons (int1) in biofilm of different DWDSs were quantified by qPCR
268
(Fig. 1). The average gene copy numbers of 16S rRNA for total bacteria were
269
(9.52±0.31), (10.9±0.33), (10.2±0.29), and (11.5±0.32) log (gene copies cm-2) in
270
biofilm
271
sulfadiazine-ciprofloxacin, respectively (Fig. 1a). The results indicated that trace level
272
of sulfadiazine and ciprofloxacin induced the increase of total bacteria in biofilms of
273
DWDSs, and the combined effects of sulfadiazine and ciprofloxacin was higher than
274
that resulted from sulfadiazine or ciprofloxacin alone.
DWDSs
with
raw
water,
sulfadiazine,
ciprofloxacin
and
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The average gene copy numbers of sul1 were (3.99±0.06), (5.40±0.12), (4.38±0.12),
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and (4.73±0.13) log (gene copies cm-2) in biofilm of DWDSs with raw water,
277
sulfadiazine, ciprofloxacin and sulfadiazine-ciprofloxacin, respectively (Fig. 1b). The
278
gene copy number of sul1 was higher than that of sul2 and sul3, and the gene copy
279
numbers of sul1, sul2 and sul3 correlated very well (r>0.98, p<0.05) (Table S5). In
280
addition, the average gene copy numbers of qnrS were (1.55±0.03), (3.34±0.05),
281
(3.88±0.04), and (3.64±0.05) log (gene copies cm-2) in biofilm of the four kinds of
282
DWDSs. The gene copy number of qnrB showed the same changes with qnrS. The
283
results indicated that the higher concentration of sulfadiazine and ciprofloxacin
284
induced the higher increase of sulfadiazine and ciprofloxacin resistance genes,
285
respectively, which suggested that the concentrations of these antibiotics were the
286
main determinants of sul1, sul2, sul3, qnrB and qnrS promotion.
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(5.41±0.15) and (5.61±0.18) log (gene copies cm-2) in biofilm of DWDSs with raw
289
water, sulfadiazine, ciprofloxacin and sulfadiazine-ciprofloxacin, respectively.
290
Ciprofloxacin induced higher increase of mexA than that caused by sulfadiazine. The
291
multiple mex efflux genes can confer bacterial resistance to nearly all the antibiotics
292
by pumping out the antibiotics (Jia et al., 2015; Tandukar et al., 2013). Therefore, the
293
combined effect of sulfadiazine and ciprofloxacin resulted in the highest promotion of
294
mexA. In addition, the gene copy number of int1 were (4.31±0.11), (4.61±0.15),
295
(4.43±0.13) and (5.17±0.16) log (gene copies cm-2) in biofilm of the four kinds of
296
DWDSs. An integron such as int1 is a typical gene capture and dissemination system,
297
which is nearly ubiquitous in various DNA materials (Chen et al., 2015; Wu et al.,
298
2016). Bacteria with int1 had a selective advantage compared to other bacteria, and
299
int1 was strictly correlated to co-selection mechanisms (Cesare et al., 2016).
300
Therefore, sulfadiazine and ciprofloxacin induced the increase of int1 obviously, and
301
the combined effect of sulfadiazine and ciprofloxacin was stronger than that induced
302
by sulfadiazine or ciprofloxacin alone.
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The gene copy numbers of 16S rRNA and int1 correlated very well (r>0.95, p<0.05)
304
(Table S5). Integrons such as int1 play a great role in the horizontal gene transfer of
305
ARGs between microbes (Gaze et al., 2011). Some studies also indicated that the shift
306
of bacterial community due to the antibiotics could promote the bacterial resistance
307
(Jia et al., 2015; Davids et al., 2017). Therefore, the promotion of the ARGs in biofilm
308
of different DWDSs, which was induced by the addition of sulfadiazine and 14
ACCEPTED MANUSCRIPT 309
ciprofloxacin, may be related with the increase of the total bacteria and the bacterial
310
community shift in biofilm.
311
3.3. Bacterial community shift in biofilm of different DWDSs Based on the sequencing analysis of 16S rRNA genes, Proteobacteria phylum
313
dominated in the biofilm bacterial communities of the four kinds of DWDSs, and its
314
relative abundance in all the biofilm bacterial communities was more than 89.7% (Fig.
315
S3a). At class level, Alphaproteobacteria was the main class in all the biofilm
316
bacterial communities (Fig. S3b). Furthermore, the dominant bacteria were compared
317
at genus level for different samples (Fig. 2). Hyphomicrobium, Sphingopyxis,
318
Sphingomonas, Rhodobacter and Parvularcula were the predominant bacterial genera
319
in biofilm bacterial communities of DWDSs with raw water. The relative abundance
320
of Sphingopyxis and Parvularcula decreased in biofilm bacterial communities of
321
DWDSs with sulfadiazine, ciprofloxacin, and sulfadiazine-ciprofloxacin, which may
322
be due to the antibiotic activity of these antibiotics. The relative abundance of
323
Rhodobacter did not take great changes in DWDSs with the addition of sulfadiazine
324
and ciprofloxacin. However, compared with the DWDSs with raw water, the relative
325
abundance of Hyphomicrobium increased from 40.3% to 46.7%, 43.3% and 49.0% in
326
DWDSs with sulfadiazine, ciprofloxacin, and sulfadiazine-ciprofloxacin, respectively.
327
Moreover, the relative abundance of Bosea, Bdellovibrio and Sphingomonas also
328
increased in biofilm bacterial communities of DWDSs with these antibiotics.
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Compared with DWDSs with raw water, the 16S rRNA for total bacteria and the
330
relative abundance of Hyphomicrobium, Bosea, Bdellovibrio and Sphingomonas 15
ACCEPTED MANUSCRIPT increased in biofilm of DWDSs with antibiotics. The results suggested that the growth
332
of these bacterial genera was contributed to the increase of total bacteria in biofilm.
333
Moreover, the relative abundance of the main bacterial genus Hyphomicrobium
334
correlated very well with the gene copy numbers of 16S rRNA and int1 (r>0.95,
335
p<0.05) (Table S5). Therefore, the growth of bacterial genus Hyphomicrobium and the
336
increase of total bacteria were related with the antibiotic resistance genes in DWDSs
337
with sulfadiazine and ciprofloxacin. Moreover, the bacterial communities shift and the
338
total bacteria increase in biofilm induced the ARGs promotion, which was also
339
consistent with other studies indicating that bacterial community shift drove antibiotic
340
resistance promotion during drinking water chlorination (Jia et al., 2015).
341
3.4. Microbial metabolism behaviors
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The protease activity was (22.1±1.95), (29.3±2.05), (40.4±3.07) and (55.8±4.01) µg
343
tyrosine liberated h-1 cm-2 biofilm in DWDSs with raw water, sulfadiazine,
344
ciprofloxacin, and sulfadiazine-ciprofloxacin, respectively (Fig. 3). Moreover, the
345
dehydrogenase activity was (18.5±1.56), (36.0±3.08), (33.0±2.87) and (55.5±3.92) µg
346
TF liberated h-1 cm-2 biofilm in the four kinds of DWDSs, respectively. The results
347
indicated that trace level of sulfadiazine and ciprofloxacin increased the activity of
348
protease and dehydrogenase, and the combined effect of sulfadiazine and
349
ciprofloxacin induced the highest increase, compared with sulfadiazine or
350
ciprofloxacin alone.
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The degradation of complex polymeric organic matter by bacteria is related with
352
enzymatic activities (Lautenschlager et al., 2014; Han et al., 2016). Specific 16
ACCEPTED MANUSCRIPT hydrolytic enzymes (protease and dehydrogenase) are important for the growth of
354
microorganisms, when they were exposed to different organic matter including
355
antibiotics (Han et al., 2016; Rai et al., 2017). When antibiotics and their chlorination
356
products were present in DWDSs, they may affect the bacterial growth because of
357
their antibacterial activity. In this condition, metabolism behaviors of bacteria may
358
change due to the microbial stress responses. Therefore, protease and dehydrogenase
359
activities were affected sharply by the presence of antibiotics in our study.
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The dehydrogenase activity correlated very well with bacterial genus Bosea (r>0.97,
361
p<0.05) (Table S5). The dehydrogenase activity also showed the same tendency with
362
the main bacterial genus Hyphomicrobium. Moreover, the dehydrogenase activity
363
correlated very well with the gene copy numbers of 16S rRAN and int1 (r>0.95,
364
p<0.05). The protease activity also correlated very well with the gene copy number of
365
mexA (r>0.97, p<0.05) (Table S5). Therefore, the enzymatic activity of biofilm
366
bacterial communities might induce the increase of total bacteria in biofilm of
367
DWDSs with sulfadiazine and ciprofloxacin, and the increase of total bacteria also
368
induced the promotion of ARGs in biofilm. Moreover, EPS are involved in
369
extracellular electron transfer, and the presence of EPS can affect the enzymatic
370
activity (Han et al., 2017). Meanwhile, microbial metabolism behaviors may also
371
influence the EPS production. Therefore, the characterization of EPS in biofilm of
372
different DWDSs was also analyzed.
373
3.5. Roles of EPS on the biofilm formation
374
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Proteins were the main compositions of EPS, and the concentration of EPS proteins 17
ACCEPTED MANUSCRIPT were (194.7±7.36), (210.4±7.58), (198.4±7.14) and (244.0±7.18) µg cm-2 in biofilm
376
of DWDSs with raw water, sulfadiazine, ciprofloxacin, sulfadiazine-ciprofloxacin,
377
respectively (Fig. 4). The results indicated that the combined effects of sulfadiazine
378
and ciprofloxacin induced the highest increase of EPS proteins, followed by
379
sulfadiazine and ciprofloxacin alone.
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Several regions on the FTIR spectra provided information regarding major
381
functional moieties of EPS (Hessler et al., 2012). FTIR spectra showed that nine
382
peaks were present in EPS (Fig. 5a). The peaks at 1049 and 1076 cm-1 were related to
383
C-O stretching of polysaccharides (Adeleye and Keller, 2016). The peak at 1248 cm-1
384
was related to P=O stretching of phospholipids or nucleic acids (Adeleye and Keller,
385
2016). The bands at 1410 cm-1 and 1452 cm-1 corresponded to C–H bending and C–N
386
stretching vibrations of proteins, respectively, which may arise from the amide II (You
387
et al., 2015). The peak bands (1600-1500 cm-1) were due to N-H bending and C-N
388
stretching vibrations in proteins (amide II band), which was very weak in EPS
389
(Adeleye and Keller, 2016). The amide I band (1700-1600 cm-1) was attributable to
390
the C=C and C=O stretching in proteins (Hessler et al., 2012; Jia et al., 2017). The
391
peaks at 2897 and 2972 cm-1 were assigned to C-H antisym and sym stretching in
392
lipids (Adeleye and Keller, 2016). Moreover, for elucidating the changes of the
393
secondary structure of proteins, the curve fitting of original infrared spectra for amide
394
I region were performed (Fig. 5b and Fig. S4). The protein secondary structures
395
included aggregated strands, β-sheet, random coil, α-helix, 3-turn helix and
396
antiparallel β-sheet (Han et al., 2017). The relative contents of these secondary
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18
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398
raw water, the relative contents of β-sheet in EPS from biofilm increased from 20.3%
399
to 25.6%, 22.1% and 27.6% in DWDSs with sulfadiazine, ciprofloxacin and
400
sulfadiazine-ciprofloxacin, respectively. The changes of the relative contents of
401
β-sheet showed the same tendency with the proteins concentration in EPS. The
402
combined effects of sulfadiazine and ciprofloxacin induced the highest increase of
403
β-sheet, followed by sulfadiazine and ciprofloxacin alone.
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As we all known, EPS played great roles on the bacterial irreversible adherence
405
onto surface to form biofilm (Xue et al., 2012; Fish et al., 2016). In our study,
406
sulfadiazine and ciprofloxacin induced more production of EPS with higher contents
407
of proteins and β-sheet. The relative abundance of Hyphomicrobium and Bdellovibrio
408
correlated very well with β-sheet (r>0.95, p<0.05) (Table S5). Moreover, the gene
409
copy numbers of 16S rRNA and int1 correlated very well with the EPS proteins and
410
β-sheet, respectively (r>0.95, p<0.05) (Table S5). The gene copy numbers of 16S
411
rRNA and int1 also showed good relationship with the enzymatic activities (r>0.95,
412
p<0.05) (Table S5). Therefore, the total bacteria exhibited the highest enzymatic
413
activities in DWDSs with sulfadiazine-ciprofloxacin and induced the highest EPS
414
production. The higher contents of proteins increased the hydrophobicity of cell
415
surfaces (Jia et al., 2017). Due to the twisted and pleated sheet structure of β-sheet,
416
large amounts of inner hydrophobic groups of amino acids were more easily to be
417
exposed and express the hydrophobic property of the bacteria (Hou et al., 2015). The
418
increased hydrophobicity of bacterial surfaces due to the higher contents of proteins
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420
pipelines to form biofilm. The results were consistent with other studies which
421
indicated that the secondary structure of protein in EPS such as β-sheet played
422
important roles in bioflocculation and adhesion (Yin et al., 2015; Jia et al., 2017).
423
Moreover, EPS can serve as a barrier to mitigate the disinfectant and antibiotics
424
intrusion into the bacteria (Han et al., 2017). Therefore, the bacterial communities
425
changed and the relative abundance of Hyphomicrobium enhanced, meanwhile, the
426
gene copy number of 16S rRNA for total bacteria also increased in biofilm of DWDSs
427
with sulfadiazine and ciprofloxacin, which induced the ARGs promotion. The
428
combined effect of sulfadiazine and ciprofloxacin was stronger than that resulted from
429
sulfadiazine and ciprofloxacin alone.
430
4. Conclusions
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Compared with DWDSs with raw water, the gene copy numbers of 16S rRNA for
432
total bacteria and the ARGs and class 1 integrons (int1) increased in DWDSs with
433
sulfadiazine and ciprofloxacin. The relative abundance of bacterial genus
434
Hyphomicrobium also enhanced. The increase of total bacteria was related to the
435
ARGs promotion. Moreover, the bacteria exhibited higher enzymatic activities and
436
produced more EPS in DWDSs with sulfadiazine and ciprofloxacin. The higher
437
contents of EPS proteins and secondary structure β-sheet promoted bacterial
438
adsorption onto surface of pipelines to form biofilm. Therefore, the total bacteria
439
increased in DWDSs with antibiotics, which also drove ARGs promotion.
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Acknowledgments This work was funded by the National Natural Science Foundation of China (Nos. 51878654,
444
(QYZDY-SSW-DQC004) and the Federal Department of Chinese Water Control and
445
Treatment (Nos. 2017ZX07108, 2017ZX07501002).
446
Declaration of interest
449
project
of
None
Chinese
Academy
of
Sciences
SC
448
the
Appendix A. Supplementary data
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51838005),
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Supplementary material related to this article can be found in the online version.
450
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Table Captions
3
Table 1. The relative content of each secondary structure in amide I region of EPS
4
proteins from biofilm bacterial communities in different drinking water distribution
5
systems (%).
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6
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1
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11
Table 1. The relative content of each secondary structure in amide I region of EPS
13
proteins from biofilm bacterial communities in different drinking water distribution
14
systems (%). protein secondary
SC
12
Raw
Two
Sulfadiazine Ciprofloxacin water
aggregated strands
7.28
9.64
8.16
5.72
β-sheet
20.3
25.6
22.1
27.6
random coil
26.7
21.5
16.6
23.6
α-helix
29.1
26.9
35.5
27.7
3-turn helix
10.8
12.4
12.8
9.13
5.74
4.03
4.87
6.22
17 18 19 20
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16
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antiparallel β-sheet 15
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2
antibiotics
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Figure Captions
3
Fig. 1. The gene copy numbers of 16S rRNA for total bacteria (a) and the antibiotic
4
resistance genes (b) in biofilm of different drinking water distribution systems. Error
5
bars represent the standard deviation from the average of three replications.
6
Fig. 2. The relative abundance of main bacterial genera in biofilm of different
7
drinking water distribution systems. Other bacterial genera, relative abundance of
8
which was lower than 1% in all the samples, were not shown.
9
Fig. 3. The enzymatic activities of protease and dehydrogenase in biofilm of different
10
drinking water distribution systems. Error bars represent the standard deviation from
11
the average of three replications.
12
Fig. 4. The proteins concentration in EPS from biofilm bacterial community of
13
different drinking water distribution systems. Error bars represent the standard
14
deviation from the average of three replications.
15
Fig. 5. The FTIR spectra of EPS from biofilm bacterial community in different
16
drinking water distribution systems (a), and the secondary structures and curve-fitted
17
amide I region (1700-1600 cm-1) of EPS in biofilm of DWDSs with raw water (b).
SC
M AN U
TE D
EP
AC C
18
RI PT
2
1
ACCEPTED MANUSCRIPT
12
(a)
10
6 4 2 0
e cin iotics water diazin floxa antib Raw Sulfa Cipro Two
RI PT
8
5 4
2 1
mexA int1
sul1
sul2
sul3 qnrB qnrS
AC C
EP
0
Raw water Sulfadiazine Ciprofloxacin Two antibiotics
TE D
3
M AN U
(b)
6
-2
Log (gene copies cm biofilm)
20
21
16S rRNA
SC
log (gene copies cm-2 biofilm)
19
22
Fig. 1. The gene copy numbers of 16S rRNA for total bacteria (a) and the antibiotic
23
resistance genes (b) in biofilm of different drinking water distribution systems. Error
24
bars represent the standard deviation from the average of three replications.
25
2
ACCEPTED MANUSCRIPT 26 27 28
RI PT
29 30
SC
31
60
Rhizobacter Parvularcula Rhodobacter Sphingomonas Bdellovibrio Bosea Methylobacterium Sphingopyxis Hyphomicrobium
50
M AN U
Relative abundance (%)
70
40 30 20 10 0
32
TE D
water azine xacin iotics Raw SulfadiCiproflTowo antib
Fig. 2. The relative abundance of main bacterial genera in biofilm of different
34
drinking water distribution systems. Other bacterial genera, relative abundance of
35
which was lower than 1% in all the samples, were not shown.
AC C
36
EP
33
3
ACCEPTED MANUSCRIPT 37 38 39
RI PT
40 41
-1
30 20 10 0
Protease
Dehydrogenase
TE D
43
40
SC
50
M AN U
Raw water Sulfadiazine Ciprofloxacin Two antibiotics
60
-2
Enzymatic activity (µg h cm biofilm)
42
Fig. 3. The enzymatic activities of protease and dehydrogenase in biofilm of different
45
drinking water distribution systems. Error bars represent the standard deviation from
46
the average of three replications.
AC C
47
EP
44
4
ACCEPTED MANUSCRIPT 48 49 50
RI PT
51 52
150 100 50
e cin iotics water diazin floxa antib Raw Sulfa Cipro Two
TE D
0
53
SC
200
M AN U
-2
Proteins (µg cm biofilm)
250
Fig. 4. The proteins concentration in EPS from biofilm bacterial community of
55
different drinking water distribution systems. Error bars represent the standard
56
deviation from the average of three replications.
AC C
57
EP
54
5
ACCEPTED MANUSCRIPT 58 59
(a) 1049
2972
1628 1410 1248 1452 1654
1076
RI PT
2897
Absorbance
Two antibiotics Sulfadiazine Ciprofloxacin
1000
1500
2000
2500
60
0.030
(b)
0.015 0.010 0.005
TE D
Absorbance
0.025 0.020
3000
M AN U
Wavenumber (cm-1)
SC
Raw water
raw data aggregated strands β-sheet random coil α-helix 3-turn helix antiparallel β-sheet
EP
0.000
1600 1620 1640 1660 1680 1700 1720 -1
61
AC C
Wavenumber (cm )
62
Fig. 5. The FTIR spectra of EPS from biofilm bacterial community in different
63
drinking water distribution systems (a), and the secondary structures and curve-fitted
64
amide I region (1700-1600 cm-1) of EPS in biofilm of DWDSs with raw water (b).
65
6
ACCEPTED MANUSCRIPT Highlights Response of biofilm to sulfadiazine and ciprofloxacin in DWDSs was studied. Bacterial community shift and total bacteria increase related to ARGs promotion.
RI PT
The bacteria exhibited higher enzymatic activity and produced more EPS. The higher contents of proteins and β-sheet in EPS promoted biofilm formation.
AC C
EP
TE D
M AN U
SC
Sulfadiazine and ciprofloxacin exhibited stronger combined effects on biofilm.