Subinhibitory Concentrations of Disinfectants Promote the Horizontal

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Sub-inhibitory Concentrations of Disinfectants Promote the Horizontal Transfer of Multidrug Resistance Genes within and across Genera Ye Zhang, April Z Gu, Miao He, Dan Li, and Jianmin Chen Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b03132 • Publication Date (Web): 05 Dec 2016 Downloaded from http://pubs.acs.org on December 10, 2016

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Environmental Science & Technology

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Sub-inhibitory Concentrations of Disinfectants Promote the Horizontal

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Transfer of Multidrug Resistance Genes within and across Genera

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Ye Zhang†, April Z. Gu‡, Miao He§, Dan Li†*, Jianmin Chen†

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† Shanghai Key Laboratory of Atmospheric Particle Pollution and Prevention (LAP3), Department of

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Environmental Science and Engineering, Fudan University, Shanghai, 200433, China.

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‡ Department of Civil and Environmental Engineering, Northeastern University, Boston, MA, 02115,

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U.S.A.

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§ Environmental simulation and pollution control (ESPC) State Key Joint Laboratory, School of

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Environment, Tsinghua University, Beijing, 100084, China.

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*Corresponding authors: D. L (Phone: +86 (021) 65642024; E-mail: [email protected]).

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ACS Paragon Plus Environment


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ABSTRACT: The greater abundances of antibiotic resistance genes (ARGs) in point-of-use tap and

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reclaimed water than that in freshly treated water raise the question whether residual disinfectants in

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distribution systems facilitate the spread of ARGs. This study investigated three widely-used

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disinfectants (free chlorine, chloramine, and hydrogen peroxide) on promoting ARGs transfer within

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Escherichia coli strains and across genera from Escherichia coli to Salmonella typhimurium. The results

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demonstrated that sub-inhibitory concentrations (lower than minimum inhibitory concentrations [MICs])

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of these disinfectants, namely 0.1–1 mg/L Cl2 for free chlorine, 0.1–1 mg/L Cl2 for chloramine, and

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0.24–3 mg/L H2O2, led to concentration-dependent increases in intra-genera conjugative transfer by 3.4–

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6.4, 1.9–7.5, and 1.4–5.4 folds compared with controls, respectively. By comparison, the inter-genera

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conjugative frequencies were slightly increased by approximately 1.4–2.3 folds compared with controls.

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However, exposure to disinfectants concentrations higher than MICs significantly suppressed

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conjugative transfer. This study provided evidences and insights into possible underlying mechanisms

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for enhanced conjugative transfer, which involved intracellular reactive oxygen species formation, SOS

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response, increased cell membrane permeability, and altered expressions of conjugation-relevant genes.

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The results suggest that certain oxidative chemicals, such as disinfectants, accelerate ARGs transfer and

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therefore justify motivations in evaluating disinfection alternatives for controlling antibiotic resistance.

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This study also triggers questions regarding the potential role of environmental chemicals in the global

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spread of antibiotic resistance.

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Keywords: Antibiotic resistance genes; conjugative transfer; horizontal transfer; reactive oxygen

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species; SOS response

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INTRODUCTION The development and spread of antibiotic resistance poses a serious public health threat on a global

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scale.1, 2 Bacteria acquire and dissemination antibiotic resistance via genetic mutations and horizontal

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transfer of antibiotic resistance genes (ARGs),2, 3 and the occurrences of antibiotic resistance have been

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traditionally considered to be caused by misuse or overuse of antibiotics in clinical and agricultural for

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humans,2 livestock,4 and aquatic products.5 Horizontal transfer of ARGs is considered as one of the

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major drivers that accelerate the development and enrichment of antibiotic resistance in the

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environment,3, 6 and it is generally associated with foreign genetic elements containing ARGs, such as

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plasmids, transposons and integrons.3, 7, 8 Usually, horizontal transfer of ARGs occurs between closely

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related species of bacteria, but it also happens across bacterial genera, occurring at a relatively low

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frequency but posing significant clinical importance.7, 9

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Some antibiotics are constantly escaping into the environment as a result of the long term and

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uncontrolled application of antibiotics, which produces an additional selection pressure for antibiotic

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resistance.10 As expected, antibiotic resistant bacteria (ARB) and ARGs have been detected frequently in

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environmental water, including municipal wastewater,11 reclaimed wastewater,12 and even drinking

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water.13 The effects of disinfection processes on the removal of ARB and ARGs in reclaimed water and

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drinking water have been investigated in the laboratory scale14, 15 and field studies.16 However, these

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results cannot confirm that disinfectants are playing a key role in the control of the occurrence and

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dissemination of antibiotic resistance in real water systems.17, 18 It was observed that although the levels

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of bacteria were significantly reduced in the finished reclaimed water and drinking water after

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disinfection treatments, the abundances of both ARB and ARGs were much higher in the point-of-use of 3



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tap and in reclaimed water than those in the freshly treated water.19 Thus, water distribution systems

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have the potential to facilitate the spread and enrichment of antibiotic resistance.

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Conventionally, residual disinfectants, such as free chlorine, chloramine, and hydrogen peroxide

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(H2O2), are applied to control the regrowth of microorganisms in both drinking and reclaimed water

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distribution pipelines.20–22 The residual disinfectants in the drinking distribution system after

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long-distance distribution, or in point-of-use of tap water, often decrease to sub-inhibitory levels or are

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even undetectable.20–23 Therefore, ARB and ARGs in disinfected drinking and reclaimed water may be

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exposed to relatively low concentrations of disinfectants for a period ranging from a few hours to a few

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days.

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Recent studies have explored the ability and the underlying mechanisms of sub-inhibitory levels

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(tens to hundreds of folds below the minimal inhibitory concentrations [MICs], also referred to as

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sub-MICs or sub-lethal concentrations) of antibiotics to induce resistant mutants and to stimulate the

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horizontal transfer of ARGs.3, 24, 25 Evidences indicated that the horizontal transfer of ARGs between

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bacteria can be induced by sub-inhibitory levels of antibiotics via broadly conserved cellular response

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pathways such as those involved in reactive oxygen species (ROS) response systems25 and the SOS

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response.3, 25 The involvements of these conserved cellular pathways and mechanisms raise important

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implications regarding the possible role of environmental chemicals other than antibiotics in the

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transmission of ARGs. A recent study of the effects of chlorine and ultraviolet irradiation (UV)

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disinfection processes on the horizontal transfer of ARGs indicated that the frequency of conjugative

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transfer was significantly increased by sub-inhibitory chlorine doses (up to 40 mg Cl2 min/L), while

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inhibitory doses of chlorine (> 80 mg Cl2 min/L) or UV (> 10 mJ/cm2) greatly suppressed the 4


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frequencies of ARG transfers.26 The role of widespread residual disinfectants in promoting ARG transfer,

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particularly at environmentally-relevant low concentrations (sub-inhibitory levels), such as those in

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water storage and distribution systems, has seldom been evaluated and warrants a more systematic

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investigation.

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In this laboratory-based study, we investigated the effects of sub-inhibitory levels of three

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widely-used disinfectants, namely free chlorine, chloramine, and H2O2, on the conjugative transfer of

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ARGs within two different Escherichia coli (E. coli) strains and across genera from E. coli to

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Salmonella Typhimurium (S. typhimurium). Free chlorine and chloramine are residual disinfectants that

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are regulated by the U.S. Environment Protection Agency (USEPA) and other agencies worldwide.21, 22

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The maximum residual disinfectant level goals are 4.0 mg/L for both chlorine and chloramine in the

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USEPA National Primary Drinking Water Regulations (Table S1), and the drinking water sanitary

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standard in China requires that residual concentrations at the end of distribution systems must be greater

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than 0.05 mg/L chorine or 0.05 mg/L chloramine (Table S2). H2O2, the simplest peroxide, is one kind of

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ROS that is widely used as a strong oxidizer and disinfectant of skin, food, and drinking water.27 We

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also systematically explored the mechanisms that underlie this process with regards to the intracellular

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ROS, the SOS response, the cell membrane permeability, and the expression of conjugation-relevant

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genes (e.g., korA, korB, trbA, trbBp, traF, trfAp, and traJ), outer membrane-encoding genes (e.g., ompA,

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ompF, and ompC), and oxidative stress regulatory genes (e.g., rpoS). To our knowledge, this is the first

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study to explore the effect and mechanisms of sub-inhibitory levels of disinfectants on the promotion of

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the horizontal transfer of ARGs within and across genera. The results of the present study will provide

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guidance for disinfection practices for water treatment and distribution systems and contribute to

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prevention of antibiotic resistance transfer.

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MATERIALS AND METHODS

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Establishment of conjugative transfer models. In order to evaluate the conjugative transfer of ARGs

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in the environment within and across genera, two conjugative transfer models were established.

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In the intra-genera conjugative transfer model, the donor E. coli S17-1 strain, containing the

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pCM184-Cm plasmid carrying ampicillin (Amp), chloromycetin (Chl), and tetracycline (Tet) resistance

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genes, was cultured in Luria–Bertani (LB) medium (tryptone, 10 g/L; yeast extract, 5 g/L; NaCl, 10 g/L;

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pH, 7.4) containing 20 mg/L Chl. The recipient E. coli K12 strain, containing the pUA139 plasmid

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carrying a kanamycin (Km) resistance gene, was cultured in LB medium containing 100 mg/L Km.

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Intra-genera transconjugants were selected on LB plates containing 10 g/L agar, 20 mg/L Chl, and 100

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mg/L Km.

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In the inter-genera conjugative transfer model, the donor E. coli S17-1 strain, containing pBHR1

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plasmid carrying the Km and Chl resistance genes, was cultured in LB medium supplemented with 20

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mg/L Chl. The recipient S. typhimurium TA1535 strain, carrying the Amp resistance gene in its

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chromosome, was cultured in LB medium supplemented with 100 mg/L Amp. Inter-genera

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transconjugants were selected on LB agar plates that were supplemented with 20 mg/L Chl and 100

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mg/L Amp.

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In order to optimize the conjugative transfer models, the cell density (104, 105, 106, 107, 108, and 109 colony-forming units (CFU)/mL), donor/recipient ratios (0.5:1, 1:1, 1.5:1, 2:1, and 3:1), mating

6


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times (4, 8, 12, and 24 h), and temperatures (25 and 37 °C) were optimized by a single-factor

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experiment.

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Specific descriptions of all the strains and plasmids used in the present study are provided in Table

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S3. All of the donor and recipient bacteria were incubated at 37 °C for 16–18 h, with shaking at 180 rpm.

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Then, these bacterial cultures were prepared and subjected to a conjugation experiment, as well as

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assays that evaluated cell membrane permeability, oxidative stress via intracellular ROS production,

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transcriptomic analysis and the mRNA expression of integron genes.

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Conjugation experiments. In order to eliminate the influence of the culture medium, overnight cultures

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of the donor and recipient bacteria were centrifuged for 5 min at 10,000 × g and 4 °C. Following

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centrifugation, the supernatants were removed, and the bacteria were re-suspended in different volumes

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of phosphate-buffered saline (PBS, pH = 7.2) to obtain different bacterial concentrations. Then, the

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donor and recipient bacteria were mixed and exposed to different sub-MIC (sub-inhibitory)

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concentrations of disinfectants, including free chlorine (0, 0.1, 0.3, 0.5, 1, and 5 mg/L as Cl2),

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chloramine (0, 0.1, 0.3, 0.5, 1, and 5 mg/L as Cl2), and H2O2 (0, 0.24, 1.2, 3, 6, and 30 mg/L). For

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comparison, conjugation experiments were also performed with inhibitory concentrations (>90 %

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growth inhibition), namely 10 mg/L for free chlorine, 10 mg/L for chloramine, and 60 mg/L for H2O2.

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The MICs (90 % growth inhibition)28 of the donor and recipient bacteria against three disinfectants were

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pre-determined based on the concentration-inhibition curves of donor (E. coli S17-1) and recipient (E.

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coli K12 and S. typhimurium) strains following treatment with free chlorine, chloramine, and H2O2 were

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evaluated, respectively (Figure S1). The Hach pocket colorimeter II kit (cat. no. 59530-00, Hach,

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Loveland, CO, USA) was used to determine the concentrations of free chlorine and chloramine. These 7



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tested concentrations of disinfectants were selected with consideration of the residual disinfectant

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requirements of drinking water regulations (Tables S1 and S2),21, 22 as well as the concentrations

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commonly occurring in drinking water and reclaimed water storage and distribution systems in previous

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reports.20, 29 Sodium hypochlorite served as free chlorine in the experiments. Chloramine was prepared

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by reacting ammonium chloride and free chlorine according to a previous method.26

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In order to simulate water distribution processes, the mixtures were incubated in dark at 25 °C for

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30 min exposure, and then sodium thiosulfate (Na2S2O3, 3.5 %) was added to each sample for

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neutralization. After the subsequent incubation at a certain temperature for several hours that are based

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on the results of the single factor experiments, the mixtures were appropriately diluted in PBS and plated

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on LB agar plates containing the appropriate antibiotics to determine and calculate the numbers of

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donors, recipients, and transconjugants as described previously. Samples that were not exposed to the

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disinfectants were processed as controls. All of the conjugation experiments were conducted at least in

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triplicate.

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Evaluation of the effects of disinfectants on cell membrane permeability using flow cytometry. A

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flow cytometry (BD FACSCalibur, BD Biosciences, Franklin Lakes, NJ, USA) approach was employed

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to evaluate the membrane permeability of the donor and recipient bacteria, as well as control cells, upon

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exposure to the disinfectants based on DNA-intercalating fluorescent dye, propidium iodide (PI) staining

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that correlate fluorescence intensity with cell membrane permeability.8, 30, 31 The detailed detection and

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data analysis processes were conducted according previous studies (Text S1).8, 31

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Assessment of oxidative stress induced by the disinfectants. To explore whether oxidative stress

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plays an important role in promoting disinfectant-induced conjugative transfer, intracellular ROS 8


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formation was determined using the fluorescent reporter dye 2’, 7’-dichlorofluorescein diacetate

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(DCFH-DA) (Invitrogen, Carlsbad, USA) and a micro-plate reader (Synergy HTMulti-Mode, BioTek,

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Winooski, VT, USA) as described previously.32 Briefly, bacterial suspensions (approximately 106–107

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CFU/mL) were stained with DCFH-DA (at a final concentration of 10 µM) for 20 min at 37 °C in the

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dark with gentle shaking. After washing twice with PBS, the bacteria were treated with different

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disinfectants as described above. Then, all the treated samples were transferred into a 96-well plate (200

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µL per well), and the fluorescence (488 nm/525 nm) intensity (FI) was measured using the microplate

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reader. The ROS production level for each disinfectant treatment was normalized to that of the control

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samples. All of the experiments were conducted at least in triplicate. The relative fold increases in ROS

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productions were calculated using eq. 1.

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Relative ROS

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production

( folds) =

FI of

treated samples

FI of

control samples

(eq. 1)

To test if the ROS formation enhances the frequency of conjugative transfer within and across

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genera, the intra-genera conjugative models were treated with 1 mg/L free chlorine, 0.1 mg/L

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chloramine, and 3 mg/L H2O2, as well as the inter-genera conjugative models were supplemented with

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0.5 mg/L free chlorine, 0.3 mg/L chloramine, and 1.2 mg/L H2O2. Meanwhile thiourea (CH4N2S, TU)

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(TCI, Tokyo, Japan), a scavenger of ROS, was added to each culture at 100 µM final concentrations.

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Then, the process of conjugative transfer was conducted, and the numbers of transconjugants were

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counted.

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Evaluation of the mRNA expression of genes responsible for conjugative transfer. Total RNA was

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isolated from the disinfectant-treated bacterial samples using RNAiso Plus (cat. no. D9108A, TaKaRa,

9



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Dalian, China). Then, the RNA was transcribed into cDNA using a reverse transcription kit (cat. no.

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2680A, TaKaRa). The expression of horizontal transfer global regulator genes (korA, korB, and trbA),

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conjugation-related genes (trbBp, trfAp, traF and traJ), outer membrane protein-encoding genes (ompA,

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ompF, and ompC), and an oxidative stress regulatory gene (rpoS) were quantified using a real-time

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polymerase chain reaction (PCR), and 16S rRNA was used as an internal control. Real-time PCR was

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carried out by using SYBR Green I (cat. no. DRR420A, TaKaRa, Dalian, China) in a real-time PCR

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instrument (CFX 96, Bio-Rad, Hercules, CA, USA).

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The primers used in present study are shown in Table S4. The real-time PCR mixtures consisted of

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5 µL of 2 × SYBR Premix Ex TaqTM (TaKaRa, Dalian, China), 0.2 µL of each primer (10 µM final

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concentrations), 1 µL of cDNA template, and 3.6 µL of distilled H2O. The thermocycling profile for the

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amplifications was 95 °C for 30 s, followed by 40 cycles of 95 °C for 45 s, 60 °C for 45 s, a melting

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curve analysis at 95 °C for 15 s, and, finally, annealing at 60 °C for 1 min. Each experiment was

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conducted at least in duplicate.

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Transcriptomic analysis of the effects of the disinfectants on the SOS response pathways. The

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effects of the disinfectants on the SOS response pathways, which were previously shown to contribute to

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antibiotic resistance,3, 24, 25 were evaluated using a green fluorescent protein (GFP)-transformed E. coli

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K12 MG1655 library (Open Biosystems, Huntsville, AL, USA) as described previously.28, 33 Each

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promoter was expressed from a low-copy plasmid, pUA66 or pUA139, which contains a Km resistance

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gene and a fast-folding form of GFP, thus enabling real-time measurement of the gene expression. For

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this particular study, 19 recombinant E. coli strains with different promoters, which control the

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expression of the SOS response pathways, were selected (Table S5). The detection processes and data 10


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analysis were performed according to previous studies (Text S2) 28, 33.

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Statistical analysis. Each experiment was conducted independently at least in triplicate. The SPSS 16.0

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(SPSS, Chicago, USA) was performed for all data analysis. Significant differences were statistically

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assessed using Analysis of variance (ANOVA) and Independent-sample t test. A value of P < 0.05 was

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considered to be significant, and a value of P < 0.01 was considered to be very significant.

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RESULTS AND DISCUSSION

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Optimization of experimental models for conjugative transfer within and across genera.

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Conjugative transfer models for evaluating intra- and inter-genera transfer were optimized, including the

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donor/recipient ratio, bacterial concentrations, mating times, and temperature. As shown in Figure 1a,

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the efficiency of spontaneous conjugative transfer was approximately 10−5 when the bacterial

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concentrations ranged from 106 to108 CFU/mL. This result is consistent with previous studies where the

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bacterial concentrations applied in conjugative transfer studies generally ranged from 106 to 109

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CFU/mL.9, 26, 34 Very few transconjugants were detected when the bacterial concentration was less than

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105 CFU/mL. As conjugation requires cell-to-cell contact to form a pilus or pores that are needed for

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plasmid transfer, higher bacterial concentrations lead to higher frequencies of cell-to-cell contacts.26

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Previous studies observed bacterial concentrations ranging from 102 to 105 CFU/mL in reclaimed

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water,35 and less than 102 CFU/mL in drinking water.19, 22, 36 However, the potential numbers of donor

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and recipient bacteria in biofilms in the storage and distribution systems of drinking and reclaimed water

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are rather high, which may imply higher frequency of conjugative transfer.37, 38 Because the goal of this

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study was to elucidate the potential and mechanism of horizontal transfer of ARGs, we selected the

11



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experimental conditions that yielded the highest transfer efficiency for further evaluations. The frequency of intra-genera conjugative transfer ranged from 1.96 × 10–6 to 5.12 × 10–6 was

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slightly influenced by the donor/recipient ratios (from 0.5:1 to 3:1), and the highest frequency of

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inter-genera conjugative transfer occurred when the donor/recipient ratio was 1.5:1 in our study (Figure

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1c). Mating time significantly influenced the frequency of intra- and inter-genera conjugative transfer,

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especial for the model from E. coli S17-1 to E. coli K12, which decreased when the mating time was

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greater than 4 h (Figure 1b and 1c). Previous studies found that the conjugative transfer of the RP4

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plasmid required 6–8 h of mating to obtain the optimal efficiencies,9, 26 which was longer than that of the

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pCM184-Cm and pBHR1 plasmids in the present study. This may be because the RP4 plasmid (60,099

225

bp) is much larger than pCM184-Cm (7625 bp) and pBHR1 (5300 bp).

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The mating temperature (25 °C and 37 °C) did not have a significant effect on the conjugative

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transfer efficiency (data not shown). Therefore, the intra- and inter-genera conjugative models were

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optimized when the bacterial concentration ranged from 108 to 109 CFU/mL, with a donor/recipient ratio

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of 1.5:1, and 4 h of mating in PBS at 25 °C. The optimized models were applied in all of the following

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experiments to investigate the effects of sub-inhibitory concentrations of the disinfectants on the

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conjugative transfer of antibiotic resistance plasmids within and across genera.

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Sub-inhibitory concentrations of disinfectants promote the conjugative transfer of ARGs within

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and across genera. To test the hypothesis that sub-inhibitory levels of disinfectants increase horizontal

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gene transfer, we evaluated both intra- and inter-genera conjugate transfer following in exposure to a

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range of sub-MIC concentrations of chlorine, chloramine and H2O2. The results showed that certain

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sub-inhibitory concentrations (lower than MICs) of these disinfectants, namely 0.1–1 mg/L Cl2 for free 12


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chlorine, 0.1–1 mg/L Cl2 for chloramine, and 0.24–3 mg/L H2O2, led to concentration-dependent

238

increases in intra-genera conjugative transfer by 3.4–6.4, 1.9–7.5, and 1.4–5.4 folds compared with

239

controls, respectively (Figure 2). These low levels of disinfectants were below the maximum levels of

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residual disinfectants in drinking water (Tables S1 and S2), which represent the levels that occur in

241

drinking water and reclaimed water storage and distribution systems.20, 29

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By comparison, the inter-genera conjugative frequencies from E. coli S17-1 to S. typhimurium were

243

only slightly increased by approximately 1.4–2.3 folds following treatment with sub-inhibitory

244

concentrations of these three disinfectants (Figure 2). For both the intra- and inter-genera conjugative

245

transfer experiments, the frequencies of conjugative transfer were suppressed when the concentrations of

246

the disinfectants increased to inhibitory levels (Figure S1 and Figure 2). These results are mainly due to

247

the inactivation of both the donor and recipient bacteria treated with high levels of disinfectants (Figure

248

S1), which is in agreement with previous studies.9, 26 These previous studies showed that decreased

249

conjugative transfer was due to the reduced numbers of donor and recipient bacteria following exposure

250

to inhibitory levels of nanoparticles of aluminum oxide9 or an ionic liquid.8

251

Mechanisms by which subinhibitory disinfectants promote intra- and inter-genera conjugative

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transfer. Disinfectants, including free chlorine, chloramine, and H2O2, can produce radicals and

253

stimulate the formation of intracellular ROS, which are highly reactive molecules interference with the

254

normal functions of bacteria during aerobic respiration.27 ROS can directly damage cell membranes and

255

DNA, and then induce the SOS response, which is a global regulatory response protecting cells from

256

DNA damage.27, 39 Previous studies revealed that sub-inhibitory concentrations of antibiotics induced

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horizontal transfer of ARGs through conserved ROS and SOS response pathways.3, 25 Reports also found 13



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that nanomaterials and ionic liquids stimulated conjugative transfer by increasing cell membrane

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permeability and affecting the expression of conjugation-related genes.8, 9 In this study, we hypothesized

260

that the generation of intracellular ROS by sub-inhibitory concentrations of the disinfectants can

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promote intra- and inter-genera conjugative transfer by inducing the SOS response, increasing cell

262

membrane permeability, and altering the expression of conjugation-related genes.

263

1. Effects of sub-inhibitory concentrations of disinfectants on ROS formation and SOS response

264

To determine the effects of the disinfectants on the levels of ROS formation in donor and recipient

265

bacteria, intracellular ROS were measured using DCFH-DA.32 The radical levels in both the donor and

266

recipient bacteria significantly increased with the increasing concentrations of disinfectants compared

267

with the control samples (Figure 3 a, b and c). H2O2 exhibited the highest oxidative capacity, while free

268

chlorine caused greater oxidative stress than chloramine.

269

To determine whether the observed increase in ROS formation in both the donor and recipient

270

bacteria was related to intra- and inter-genera conjugative transfer, the correlation between conjugative

271

transfer and ROS production was determined (Figure S2). Interestingly, it is notable that high levels

272

(>10 folds increases) of ROS formation suppressed conjugative transfer, while medium levels

273

(approximately 1.5–5 folds increases) of ROS formation promoted conjugative transfer (Figure 3 and

274

Figure S2). This may be due to that the high levels of ROS damaged bacterial components, while

275

moderate ROS levels probably affected membrane permeability that plays a vital role in the transfer of

276

plasmids encoding multiple antibiotic resistances, and increased the expression of genes that are

277

involved in conjugative transfer.9, 25, 27

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The addition of thiourea, which is widely considered as a scavenger of ROS, significantly reduced 14


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the conjugative transfer frequency (Figure S3). This result further indicated that the level of ROS

280

production induced by disinfectants is one of important parameters for ARGs transfer.

281

Previous studies indicated that antibiotics could stimulate horizontal transfer of ARGs, which is

282

mediated by the induction of SOS response.3, 25 For example, antibiotic-stimulated SOS induction can

283

promote the transmission of ARGs, as exemplified by the spread of integrative conjugative elements

284

throughout populations of Vibrio cholerae.25 The three disinfectants studied here have been shown to

285

cause oxidative stress and DNA damage.27, 40 Our transcriptional analysis using the promoter insertion

286

library also demonstrated that sub-inhibitory levels of these three disinfectants induced significant

287

expression changes in SOS response-related DNA damage and repair genes (i.e., recA, polB, uvrD,

288

umuD, ssb, and ada) (Table S5).33

289

We also quantified rpoS gene expression using a quantitative real-time PCR, and the results

290

indicated that rpoS gene expression was significantly induced by these three disinfectants compared

291

with the control samples (Figure 3 d, e and f). RpoS is a stress-response sigma factor, and it is known to

292

play a role in promoting survival in response to exposure to multidrug resistance and various

293

environmental stress conditions.3, 41 A previous study found that RpoS positively regulates the small

294

RNA (sRNA) SdsR, and the up-regulation of rpoS expression led to elevated levels of sRNA that bound

295

to and repressed the translation of mutS mRNA.41 As a result, cells became depleted for the MutS

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protein, which has a central role in the repair of replication errors. ROS formation and oxidative stress

297

can induce the RpoS regulon (Figure 3), and subsequently improve the rates of intra-chromosomal

298

recombination3, which may cause more severe cell damage, such as the increased membrane

299

permeability and the altered expression of some genes. Therefore, the present results suggest that 15



300

sub-inhibitory concentrations of these three disinfectants likely stimulated the conjugative transfer of

301

ARGs via ROS formation-mediated SOS induction.

302

2. Effects of sub-inhibitory concentrations of disinfectants on cell membrane permeability

303

Page 16 of 41

The changes in membrane permeability of both the donor and recipient bacteria treated with the

304

disinfectants, as well as untreated bacteria, were evaluated with PI staining followed by flow cytometry.

305

The results showed a concentration-dependent increase in the percentage of PI-positive cells (indicating

306

increased membrane permeability) with increasing sub-inhibitory concentrations of the disinfectants

307

(Table S6 and Figure S4). However, when the levels of disinfectants were greater than inhibitory

308

concentration, for example, 10 mg/L for free chlorine, 10 mg/L for chloramine and 60 mg/L for H2O2,

309

conjugative transfer was repressed, likely due to the fatal membrane damage and cytotoxicity (Table S6).

310

No significant differences in cell permeability were observed between donor and recipient cells when

311

the cells were exposed to the same disinfectants for both intra- and inter-genera conjugative transfers

312

(Table S6 and Figure S4).

313

The bacterial outer membrane is an important barrier against horizontal gene transfer from donor to

314

recipient bacteria. During conjugation, plasmid DNA crosses the cell membrane of the donor, and then it

315

is transferred to the recipient.42 Several previous studies demonstrated that many chemicals, such as

316

antibiotics,43 chloramine,26 nanomaterials,9 and an ionic liquid,8 affect bacterial membrane permeability

317

by changing the membrane composition and structure. In addition, a number of researchers have

318

proposed that some environmental pollutants damage cell membranes by means of inducing oxidative

319

stress and ROS, as well as enhancing the expression of regulatory genes via the error-prone SOS

320

response.3, 25 16


Page 17 of 41


321

3. Effects of sub-inhibitory concentrations of disinfectants on the expression of outer membrane

322

protein-encoding genes

323

Bacteria adapt their membrane permeability by modulating the expression of outer membrane

324

proteins (OMPs). Several classical OMPs, including OmpA (34 kDa), OmpF (35 kDa), and OmpC (36

325

kDa), are responsible for the membrane permeability of donor and recipient bacteria. And they play

326

important roles in pore formation and horizontal gene transfer.44 As shown in Figure 4, the mRNA

327

expression of the ompA, ompF, and ompC genes significantly increased by approximately 1.8–7.8 folds

328

for intra-genera and 1.1–3.9 folds for inter-genera conjugative transfer following exposure to the three

329

disinfectants, respectively. A previous study confirmed that the ompA gene is primarily regulated at the

330

post-transcriptional level,45 and the post-transcriptional levels of ompA expression in both E. coli and S.

331

typhimurium were enhanced in the present study (Figure 4 and Figure S5). Similarly, Wang et al.46

332

revealed that ompA mRNA expression was up-regulated in E. coli HB101 and S. typhimurium following

333

treatment with an ionic liquid (1-ethyl-3-methylimidazolium chloride).

334

Gram-negative bacteria are capable of adapting to environmental stress, such as osmolality, pH,

335

temperature, and starvation, by changing the composition of OmpA, OmpC, and OmpF in the outer

336

membrane.47 It has been demonstrated that OmpA and OmpC are associated with the membrane

337

transport of genetic information between the inside of the cell and the external environment.48 The

338

increased formation of ROS in the donors and recipients, which was induced by the disinfectants, was

339

suspected to influence the OMPs in the cell membrane. The enhanced expression of membrane proteins

340

plays a vital role in forming outer membrane pores and augmenting membrane permeability, which may

341

pose a potential risk for antibiotic resistance dissemination via the horizontal transfer of ARGs. 17



Page 18 of 41

342

Interestingly, the omp genes and rpoS seem to be induced following similar patterns, which may due to

343

the ROS production in the whole conjugative process.

344

4. Effects of sub-inhibitory concentrations of the disinfectants on the expression of conjugative

345

transfer-related genes

346

E. coli S17-1, carrying the transfer gene of the broad-host-range RP4 plasmid integrated into its

347

chromosome,49 was used as the donor strain in present study. Conjugative transfer requires the formation

348

of conjugation bridges between the donor and recipient bacteria, which requires the regulations of the

349

global regulator genes, mating pair formation (Mpf) system genes, and plasmid transfer and replication

350

(Dtr) system genes.49, 50 In present study, we firstly determined whether sub-inhibitory levels of

351

disinfectants enhanced conjugative transfer by regulating the expression of conjugation-related genes.

352

The results showed that three disinfectants at sub-inhibitory concentrations statistically increased

353

conjugative transfer within and across genera, significantly repressed the mRNA expression of global

354

regulatory genes (korA, korB, and trbA) that are involved in conjugative transfer (Figure 5 and Figure

355

S6). Particularly, compared with the untreated control samples, the mRNA expression levels of the

356

global regulatory gene korA decreased by approximately 93.8 %, 43.3 %, and 93.8 % following

357

treatment with 1 mg/L free chlorine, 0.1 mg/L chloramine, and 3 mg/L H2O2, respectively, during

358

intra-genera conjugative transfer (Figure 5 and Figure S6). The expression levels of the global regulator

359

genes korB and trbA also decreased significantly during both intra- and inter-genera conjugative transfer

360

(Figure 5 and Figure S6). The decreased expression of global regulator genes plays an important role in

361

activating the Mpf system, which serves as the secretion machinery for the conjugative transfer of

362

plasmids.51 The repression of the korA and korB genes significantly induces the expression of the trfAp 18


Page 19 of 41


363

promoter, and the repression of the korB and trbA genes significantly induces the expression of the

364

trbBp promoter, which promotes the conjugative transfer of plasmid DNA.52

365

Additionally, increasing the mRNA expression of Mpf genes (trbBp and traF) facilitates the

366

formation of conjugants.53 The expression of trbBp gene following exposure to these three disinfectants

367

increased significantly during intra-genera conjugative transfer; however, it only increased slightly

368

during inter-genera conjugative transfer (Figure 5 and Figure S7). We also observed that the expression

369

of traF during intra-genera mating increased following exposure to sub-inhibitory concentrations of the

370

disinfectants (Figure 5). This was more pronounced for the chloramine treatment, which increased traF

371

expression by up to 5.8 folds compared with the control groups. Additionally, a previous study showed

372

that for intraspecific E. coli mating, the traF gene is essential for forming the mating bridge that serves

373

as a membrane-associated channel for the transmission of single-stranded DNA.54

374

Previous studies proved that the expression of Dtr system genes is positively correlated to

375

conjugative transfer.50, 46 Our study confirmed that sub-inhibitory levels of the disinfectants promoted

376

horizontal transfer by enhancing of the expression trfAp and traJ genes (Figure 5 and Figure S7). The

377

traJ gene is responsible for the formation of the relaxosome, which triggers a specific nick in circular

378

plasmids.55 Sub-inhibitory of these three disinfectants significantly increased traJ gene expression by

379

3.6–25.9 folds during intra-genera conjugative transfer, however, they induced less increases (2.4–5.6

380

folds) on traJ expression during inter-genera conjugative transfer (Figure 5). The significantly higher

381

expression level of the Mpf (trbBp and traF) and Dtr (trfAp and traJ) genes during intra-species

382

conjugative transfer explains why the intra-genera conjugative transfer was much higher than the

383

inter-genera conjugative transfer. 19



384

Page 20 of 41

IMPLICATIONS

385

Disinfectants, such as free chlorine, chloramine and hydrogen peroxide, which are widely used to

386

control the abundance and regrowth of microorganisms in water distribution systems and in swimming

387

pools, pose widespread and persistent exposure for both microorganisms and humans.20, 56 To our

388

knowledge, the present study is the first to demonstrate that sub-inhibitory concentrations of these

389

commonly used disinfectants promote the spread of ARGs mediated by the conjugative transfer of

390

plasmids between E. coli strains, as well as between E. coli and S. typhimurium strains, two

391

opportunistic pathogens that widely exist in the environment. The results imply that sub-inhibitory

392

concentrations of these disinfectants in water systems increase the risk of antibiotic resistance, and they

393

also suggest that the application of these disinfectants should be carefully evaluated. Further researches

394

on improving the disinfection methods to control ARB and ARGs are urgently needed.

395

Importantly, this investigation reveals potential mechanisms involved in promoting conjugative

396

transfer following exposure to sub-inhibitory concentrations of oxidative chemicals such as these

397

disinfectants. These mechanisms include intracellular ROS formation, SOS response, increase in cell

398

membrane permeability, and regulation of conjugation-relevant genes that comprise global regulator

399

genes, Mpf system genes, and Dtr system genes (Figure 6). The results of this study, consistent with

400

previous studies, suggest that environmental chemicals can stimulate horizontal transfer by producing

401

ROS and affecting the cellular SOS response pathways, which consequently impact horizontal transfer

402

and recombination of ARGs (Figure 6).3, 24, 25 The results imply that chemicals, such as disinfectants and

403

other chemicals, via mechanisms involving oxidative stress, SOS response and membrane permeability

20


Page 21 of 41


404

changes under sub-cytotoxic conditions, can potentially stimulate the spread of ARGs, thereby causing

405

serious threats to public health.

406

This study, as well as previous studies8, 9 that reported increased horizontal transfer of ARGs by

407

non-antibiotic chemicals, raises an intriguing and profound question regarding the roles of varying

408

concentrations and mixtures of environmental chemicals in the dissemination of antibiotic resistance in

409

the environment (Figure 6). Sub-inhibitory levels of disinfectants exist not only in drinking and

410

reclaimed water systems20–22, 29 and swimming pools,56 but also in food production processes and

411

healthcare facilities.27 Our results suggest that relatively low levels of disinfectants and oxidants in

412

environments and foods can enhance the spread of ARGs, thereby contributing to the emergence and

413

transmission of antibiotic-resistant, disease-causing pathogens.

414

ASSOCIATED CONTENT

415

Supporting Information. Text S1 describes the evaluation of the cell membrane permeability using

416

flow cytometry and Text S2 describes the transcriptomic analysis of impact of disinfectants on the SOS

417

response pathways. Tables summarizing of the residual disinfectants in drinking water regulated by U.S

418

Environmental Protection Agency (Table S1) and China (Table S2), descripting of the strains, plasmids

419

and primer sequences used in this study (Table S3 and S4), and summarizing expression changes in

420

genes involved in the SOS (Table S5) and cell membrane permeability by disinfectants (Table S6).

421

Figures descripting inactivation curves of donor and recipient bacteria by three disinfectants (Figure S1),

422

correlation between conjugative transfer and intracellular ROS formation (Figure S2), effects of

423

scavenging ROS on conjugative transfer of multidrug resistance genes (Figure S3), cell membrane

21



Page 22 of 41

424

permeability of bacteria treated with the disinfectants (Figure S4), mRNA expression levels of the OMPs

425

genes (Figure S5), and mRNA expression levels of genes involved in conjugative transfer exposure to

426

the disinfectants (Figure S6 and S7). This material is available free of charge via the Internet at

427

http://pubs.acs.org.

428

AUTHOR INFORMATION

429

Corresponding Author

430

Correspondence and requests for materials should be addressed to D. L. Phone: +86 (021)

431

65642024; E-mail: [email protected].

432

Notes

433

The authors declare no competing financial interest.

434 435

ACKNOWLEDGEMENTS The content is solely the responsibility of the authors. This study was supported by grants from the

436

China National Natural Science Foundation (No. 21477024 and 21527814), United States National

437

Science Foundation (NSF, CAREER CBET-0953633 and CBET-1440764), National Institute of

438

Environmental Health Sciences (NIEHS) (PROTECT P42ES017198 and CRECE P50ES026049).

22


Page 23 of 41


439

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584

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Page 31 of 41


FIGURES 1.0E-04

(a) from FromE.coli E. coliS17-1 S17-1 to coli K12 to E. E.coli K12 FromE.coli E. coliS17-1 S17-1 to typhimurium from to S. Salmonella

1.0E-03

1.0E-05

1.0E-04

1.0E-06

1.0E-05

1.0E-07

1.0E-06

Frequency of conjugative transfer across genera

Frequency of conjugative transfer within genera

1.0E-02

1.0E-08 8 9 106 107 108 109 106 107 10 10 Mating bacterial concentration (CFU/mL)

Frequency of conjugative transfer

1.0E-03

(b)

4h

8h

12h

24h

1.0E-04

1.0E-05

1.0E-06

1.0E-07 0.5:1

1:1

1.5:1

2:1

3:1

E. coli S17-1 / E. coli K12 ratio

Frequency of conjugative transfer

1.0E-04

(c)

4h

8h

12h

24h

1.0E-05

1.0E-06

1.0E-07 0.5:1

1:1 1.5:1 2:1 3:1 E. coli S17-1 / to S. typhimurium ratio

31



Page 32 of 41

Figure 1. Effects of initial bacterial concentration, donor/recipient ratio and mating time on the conjugative transfer within genera (from E. coli S17-1 to E. coli K12), and across genera (from E. coli S17-1 to S. typhimurium). (a): The effect of mating bacterial concentration on the conjugative transfer within and across genera at 4-h mating time. The mating bacterial concentration had no significant effect on intra- and inter-genera transfer (ANOVA, P > 0.05). (b): The effect of donor/recipient ratio and mating time on the conjugative transfer from E. coli S17-1 to E. coli K12. The mating time had significant effects on intra-genera transfer (ANOVA, P < 0.05), and the donor/recipient ratio had no significant effects (ANOVA, P > 0.05). (c): The effect of donor/recipient ratio and mating time on the conjugative transfer from E. coli S17-1 to S. typhimurium. The mating time and the donor/recipient ratio had significant effects on inter-genera transfer (ANOVA, P < 0.05).

32


Page 33 of 41


Fold changes of transconjugants

12

(a)

form K12 FromE.coli E. coliS17-1 S17-1totoE.coli E coli K12 FromE.coli E. coliS17-1 S17-1totoSalmonella S. typhimuriuma from

10

*

8

* *

6 *

4 *

2

**

0 0


12

(b)

0.1 0.3 0.5 1 5 Free chlorine concentration (mg/L) *

10 8 6

*

4 2

*

*

*

* *

* ** **

0 0


10

8

0.1 0.3 0.5 1 5 Chloramine concentration (mg/L)

10

(c)

7

*

6 5 4 3 ** *

2

**

** *

1 0 0

0.24

1.2 3 6 H2O2 concentration (mg/L)

30

60

33



Page 34 of 41

Figure 2. Effects of free chlorine (a), chloramine (b) and H2O2 (c) on the conjugative transfer within and across genera. No transconjugants were detected by exposure to 10 mg/L free chlorine, 10 mg/L chloramine and 60 mg/L H2O2. All disinfectants had significant effects on the intra- and inter-genera conjugative transfer of ARGs (ANOVA, P < 0.05); significant differences between individual disinfectant treated groups and the control (0 mg/L of disinfectants) were tested with Independent-sample t test, and shown with * (P < 0.05), ** (P < 0.01).

34


(a)

Donor Donor (E. (E. coli coli S17-1) S17-1) Recipient(E. (E coli coliK12) K12) Recipient Recipient (S. typhimuriuma) Recipient (Salmonella)

14 12

**

** **

10 8 6

**

4 2

**

*

**

**

**

**

**

**

**

0 0

Relative ROS production folds

4

0.1 0.3 0.5 1 Free chlorine concentration (mg/L)

5

(b) **

3

** **

**

** **

2 **

1

** ** **

** **

** **

**

0 0


60

0.1 0.3 0.5 1 Chloramine concentration (mg/L)

5

(c)

**

50 40 ** **

30 **

20 10

** ** **

**

**

**

**

**

**

** **

0 0

0.24 1.2 3 6 H2O2 concentration (mg/L)

30

rpoS expression levels (copies/16S rRNA copies)


16




Page 35 of 41

2.0E-03

(d)

FromE.coli E. coliS17-1 S17-1totoE.coli E. coli K12 from K12 FromE.coli E. coliS17-1 S17-1totoSalmonella S. typhimuriuma from **

1.5E-03

1.0E-03

*

5.0E-04

*

**

0.0E+00 0 2.0E-03

0.1 0.3 0.5 1 Free chlorine concentration (mg/L)

5

(e) **

1.5E-03

1.0E-03 *

5.0E-04

**

** ** **

0.0E+00 0 2.0E-03

0.1 0.3 0.5 1 Chloramine concentration (mg/L)

(f)

5

*

1.5E-03

1.0E-03

5.0E-04

*

**

*

*

0.0E+00 0

0.24 1.2 3 6 H2O2 concentration (mg/L)

30

Figure 3. Reactive oxygen species (ROS) production (folds) in donor and recipient bacteria induced by free chlorine (a), chloramine (b) and H2O2 (c). All disinfectants had significant effects on the production of ROS in the donor and recipient bacteria (ANOVA, P < 0.05); significant differences in ROS production between individual disinfectant treated groups and the control (0 mg/L of disinfectants)

35



Page 36 of 41

were tested with Independent-sample t test, and shown with * (P < 0.05) and ** (P < 0.01). The mRNA expression levels of oxidative stress-regulatory gene rpoS in the mating pairs within genera (from E. coli S17-1 to E. coli K12) and across genera (from E. coli S17-1 to S. typhimurium) stimulated by free chlorine (d), chloramine (e) and H2O2 (f). All disinfectants had significant effects on the rpoS gene expression in intra- and inter-genera transfer (ANOVA, P < 0.05); significant differences in the rpoS gene expression levels between individual disinfectant treated groups and the control (0 mg/L of disinfectants) were tested with Independent-sample t test, and shown with * (P < 0.05) and ** (P < 0.01).

36


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2.0E-03 1.5E-03

Outer membrane protein gene expression levels (copies/16S rRNA copies)

3.2E-03

ompA

(a) **

(b)

ompA

*

2.0E-03

2.4E-03

1.5E-03

1.6E-03

1.0E-03

ompA

(c) ** *

1.0E-03

**

*

5.0E-04

*

8.0E-04

5.0E-04

**

*

*

*

0.0E+00 3.6E-03

0

(d)

0.1

0.3

0.5

1

5

ompF

0.0E+00 2.8E-03

0

0.1

(e)

0.3

0.5

1

5

ompF

**

0

(f)

0.24

1.2

3

6

30

ompF

** **

2.4E-03

2.1E-03

2.7E-03

0.0E+00 3.2E-03

**

**

9.0E-04

* *

0.0E+00 3.2E-03

0

(g)

0.1

0.3

0.5

1

5

ompC

**

1.6E-03

0.0E+00 2.8E-03

*

0

0.1

0.3

0.5

1

5

8.0E-04

**

0

(h)

0.1

0.3

0.5

1

5

ompC

**

0.0E+00 2.8E-03

2.1E-03

2.1E-03

1.4E-03

1.4E-03 *

7.0E-04

**

**

**

*

**

**

*

**

*

**

8.0E-04

**

**

7.0E-04

**

2.4E-03

0.0E+00

1.6E-03

1.4E-03

1.8E-03

0.0E+00

Free chlorine concentration (mg/L)

0

0.1

*

**

**

0.3

*

0.24

1.2

3

6

30

ompC

*

*

*

7.0E-04

* *

** 0.5

1

5

0.0E+00

Chloramine concentration (mg/L)

From E. coli S17-1 to E. coli K12

0

(i)

0

0.24

1.2

3

6

30

H2O2 concentration (mg/L)

From E. coli S17-1 to S. typhimuriuma

Figure 4. Effects of free chlorine (a, d, g), chloramine (b, e, h) and H2O2 (c, f, i) on the mRNA expression levels of outer membrane protein genes (ompA, ompF and ompC) in the mating pairs within genera (from E. coli S17-1 to E. coli K12) and across genera (from E. coli S17-1 to S. typhimurium). Significant differences in the gene expression levels between individual disinfectant treated groups and the control (0 mg/L of disinfectants) were tested with Independent-sample t test, and shown with * (P < 0.05) and ** (P < 0.01).

37


Gene expression levels (copies/16S rRNA copies)




3.0E-03

(a)

Within-genera control Intra-genera control Within-genera mg/Lfree Free chlorine Intra-genera 1 1mg/L chlorine Cross-genera control Inter-genera control Inter-genera 0.5 Cross-genera 0.5 mg/L mg/Lfree Freechlorine chlorine

2.5E-03 2.0E-03

**

1.5E-03 1.0E-03 **

5.0E-04 **

0.0E+00

*

korA 8.0E-03

**

**

korB korB

(b)

** *

**

trbA trbBp trbB trfAp trfA trbA Free chlorine

*

** **

1.0E-03

**

5.0E-04 *

**

0.0E+00 korA

korB korB

(c)

**

*

**

**

*

trbA trbA trbBp trbB trfAp trfA Chloramine

traF traF

traJ traJ

Within-genera control Intra-genera control Intra-genera 3 3mg/L Within-genera mg/LHH2O2 2O2 Cross-genera Inter-genera control Cross-genera H2O2 Inter-genera 1.2 mg/L H 2O2

2.4E-03 1.6E-03 1.2E-03 8.0E-04

**

*

**

8.0E-04 4.0E-04

traJ traJ

**

2.0E-03

3.2E-03

traF traF

Within-genera control Intra-genera control Within-genera 0.1 mg/L chloramine Intra-genera 0.1 mg/L chloramine Inter-genera control Cross-genera control Inter-genera 0.3 Cross-genera 0.3mg/L mg/Lchloramine chloramine

4.0E-03

0.0E+00 1.5E-03

**

** **

0.0E+00 korA

korB korB

**

**

trbA trbA

trbBp trbB H2O2

trfAp trfA

**

traF traF

**

traJ traJ

38


Page 38 of 41

Page 39 of 41


Figure 5. Effects of free chlorine (a), chloramine (b) and H2O2 (c) on the mRNA expression levels of conjugation-relevant genes (korA, korB, trbA, trbBp, traF, trfAp and traJ) in the mating pairs within genera (from E. coli S17-1 to E. coli K12) and across genera (from E. coli S17-1 to S. typhimurium). Significant differences in the gene expression levels between individual disinfectant treated groups and the control (0 mg/L of disinfectants) were tested with Independent-sample t test, and shown with * (P < 0.05) and ** (P < 0.01).

39



Page 40 of 41

Possible mechanisms involved in promoting horizontal transfer of ARGs at sub-inhibitory concentrations by certain oxidative chemicals (e.g., disinfectants, antibiotics, nanoalumina ……)

Reactive oxygen species (ROS) formation

Cell membrane !  !  ! 

General diffusion porins Outer membrane barrier Secretion systems

Promotion of expression of conjugation-related genes

SOS response !  ! 

! 

RpoS induction Promotion of conjugational recombination Promotion of ARGs’ expression

!  !  ! 

Global regulator genes Mating pair formation (Mpf) system genes Plasmid transfer and replication (Dtr) system genes

Promotion the horizontal transfer of multi-antibiotic resistance genes within and across genera

Figure 6. Schematic of possible mechanisms involved in promoting horizontal transfer of antibiotic resistance genes by certain oxidative chemicals that lead to ROS formation.

40


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TOC/Abstract art:

41


Subinhibitory Concentrations of Disinfectants Promote the Horizontal

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