Phenotype and RNA-seq-Based transcriptome profiling of Staphylococcus aureus biofilms in response to tea tree oil

Accepted Manuscript Phenotype and RNA-seq-Based transcriptome profiling of Staphylococcus aureus biofilms in response to tea tree oil Xingchen Zhao, Zonghui Liu, Zuojia Liu, Rizeng Meng, Ce Shi, Xiangrong Chen, Xiujuan Bu, Na Guo PII:

S0882-4010(17)30401-1

DOI:

10.1016/j.micpath.2018.07.027

Reference:

YMPAT 3067

To appear in:

Microbial Pathogenesis

Received Date: 12 April 2017 Revised Date:

6 March 2018

Accepted Date: 20 July 2018

Please cite this article as: Zhao X, Liu Z, Liu Z, Meng R, Shi C, Chen X, Bu X, Guo N, Phenotype and RNA-seq-Based transcriptome profiling of Staphylococcus aureus biofilms in response to tea tree oil, Microbial Pathogenesis (2018), doi: 10.1016/j.micpath.2018.07.027. 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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Phenotype and RNA-seq-Based transcriptome profiling of Staphylococcus aureus biofilms in response

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to tea tree oil

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Xingchen Zhaob, Zonghui Liua , Zuojia Liuc, Rizeng Mengd, Ce Shia, Xiangrong Chena, Xiujuan Bu a,

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Na Guoa#

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a Department of Food Quality and Safety, College of Food Science and Engineering, Jilin University,

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130062, China

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b Department of Food Quality and Safety, School of Pharmaceutics and Food Science, Tonghua

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Normal University, 134000, China

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c State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry,

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Chinese Academy of Sciences, Changchun, Jilin, China

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d Jilin Entry-Exit Inspection and Quarantine Bureau, Changchun, 130062, Chinad.

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#Address correspondence to Na Guo, [email protected].

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Xingchen Zhao and Zonghui Liu contributed equally to this work

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This study depressed phenotype and expression profiles of S. aureus biofilm in the presence of TTO.

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ACCEPTED MANUSCRIPT ABSTRACT

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Staphylococcus aureus (S. aureus) is a Gram-positive bacterium that causes a wide range of diseases,

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including food poisoning. Tea tree oil (TTO), an essential oil distilled from Melaleuca alternifolia, is

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well-known for its antibacterial activities. TTO effectively inhibited all 19 tested strains of S. aureus

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biofilm and planktonic cells. Phenotype analyses of S. aureus biofilm cells exposed to TTO were

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performed by biofilm adhesion assays, eDNA detection and PIA release. RNA sequencing (RNA-seq)

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was used in our study to elucidate the mechanism of TTO as a potential antibacterial agent to evaluate

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differentially expressed genes (DEGs) and the functional network in S. aureus ATCC 29213 biofilms.

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TTO significantly changed (greater than a 2- or less than a 2-fold change) the expression of 304 genes

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in S. aureus contained in biofilms. The levels of genes related to the glycine, serine and threonine

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metabolism pathway, purine metabolism pathway, pyrimidine metabolism pathway and amino acid

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biosynthesis pathway were dramatically changed in the biofilm exposed to TTO. Furthermore, the

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expression changes identified by RNA-seq analysis were verified by real-time RT-PCR. To the best of

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our knowledge, this research is the first study to report the phenotype and expression profiles of S.

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aureus in biofilms exposed to TTO.

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Keywords Staphylococcus aureus · Tea tree oil · Biofilm · RNA sequencing

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1. Introduction Staphylococcus aureus (S. aureus) is a Gram-positive bacterium, which is considered one of the

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most important human pathogens [1], and it is one of the major foodborne and iatrogenic pathogens

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involved in a variety of diseases. A biofilm is a complex matrix produced by microorganisms in which

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cells bind to each other and link to a biotic or abiotic surface [2]. Antimicrobial resistance has become a

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highly concerning risk factor for human health worldwide. Microbial biofilms are resistant to

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antibiotics, and they play a decisive role in some persistent and chronic bacterial infections [3]. It has

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been reported that biofilms supply bacteria with an effective barrier against host immune cells [4].

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Bacteria in biofilms are disparate in phenotypic characteristics and gene expression, and they are more

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resistant to antibiotics than planktonic cells in suspension [5, 6]. Previous studies have reported that

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staphylococcal biofilms are a type of extracellular polysaccharide substance consisting of

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polysaccharide intercellular adhesion (PIA), extracellular DNA (eDNA), protein and cellular debris [7].

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eDNA exists in S. aureus biofilms and provides strength to the biofilm matrix. However, the

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mechanism of S. aureus biofilm formation is unknown.

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Essential oils (EOs) are antiseptic substances distilled from plants, and interest in EOs has

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increased in the past few years. EOs can inhibit bacterial growth by targeting the membrane and

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cytoplasm, and they can change the entire morphology of the cells in some situations [8]. Tea tree oil

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(TTO) is a type of essential oil obtained from Melaleuca alternifolia. TTO is well known for its

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effective broad spectrum activities as a topical antibacterial agent. TTO has been reported to inhibit

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bacteria at 0.002–2% and fungicide at 0.004–0.25%, and it is also an anti-inflammatory agent (≤

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0.125%) in vitro [9, 10].

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Transcriptome profiling allows the broad mapping of molecular constituents in cells, leading to

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ACCEPTED MANUSCRIPT hypotheses for the potential mechanisms of physiological and pathological conditions [11]. Over the

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past decade, RNA sequencing (RNA-seq) has become a powerful and cost-efficient tool for

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transcriptome analysis [12], and it has replaced microarrays as the preferred technique for gene

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expression analysis. In contrast to microarrays, RNA-seq has a larger range and is more sensitive and

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accurate. RNA-seq has accelerated studies to enhance our comprehension of the complexity of gene

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expression, regulation and networks [13]. A common purpose of RNA-seq is to identify DEGs between

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two or more sample groups [12].

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However, to our knowledge, no studies have used RNA-seq technology to elucidate the

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mechanism of TTO as a potential antibacterial agent to evaluate DEGs and functional network analysis

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in S. aureus biofilms and planktonic populations. Our study provides insight into the novel genes that

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may be of vital importance in biofilm formation and the mechanism of TTO effect on S. aureus

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biofilms and planktonic populations.

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2. Materials and methods

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2.1 Bacterial strain and preparation of media

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S. aureus ATCC 29213 was obtained from the China Medical Culture Collection (CMCC) Center.

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Eighteen food-borne isolates of S. aureus were obtained from the Jilin Entry and Exit Inspection and

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Quarantine Bureau. Mueller-Hinton broth II (MHB II) and Mueller-Hinton agar (MHA) were

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purchased from BD (Biosciences, Inc., Sparks, USA). TTO was obtained from Nanjing Chemlin

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Chemical Industry Co., Ltd. (Nanjing, China). Terpinen-4-ol (35-44 %), ߛ-terpinene (10-28%),

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ߙ-terpinen (5-13%), terpinolene (1.5-5%) and ߙ-terpineol (1.5–8%) are the main components of

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components of our TTO sample.

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2.2 Testing planktonic and biofilm antimicrobial susceptibility

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ACCEPTED MANUSCRIPT The minimum inhibitory concentration (MIC) analysis was performed in MHB in triplicate via

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broth microdilution techniques according to Clinical and Laboratory Standard Institute guidelines [14].

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The MICs were defined as the lowest antimicrobial concentration that inhibited >90% of growth by

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visual reading. The minimum bactericidal concentrations (MBCs) were identified as the lowest

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concentration demonstrating no microbial growth [15].

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Minimum biofilm inhibition concentration (MBIC) analysis was performed as previously

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described [16]. The MBIC was determined as the lowest concentration to show growth below or equal

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to that of the control. The minimum biofilm bactericidal concentration (MBBC) was identified as the

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lowest concentration causing no bacterial growth [15,16].

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2.3 Biofilm adhesion assays

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Biofilm adhesion was examined via a crystal violet staining assay and confocal laser scanning

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microscopy (CLSM). The crystal violet staining assay was based on a previously reported method [16].

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The TTO concentrations tested were 1, 2 and 4 mg/ml. Briefly, a static biofilm assay was performed in

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96-well plates. Biofilms were covered with crystal violet stain dissolved in cold acetic acid, and

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measurements at OD595 were recorded.

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CLSM was performed as previously reported [16]. A pre-established biofilm was stained with a

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LIVE/DEAD BacLight Bacterial Viability kit (Invitrogen Molecular Probes, Carlsbad, USA) following

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the manufacturer’s instructions. CLSM images were captured using an Olympus FV1000 confocal laser

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scanning microscope (Olympus, Tokyo, Japan) with a × 40 objective lens. Image analyses and export

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were performed with FluoView version 1.7.3.0.

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2.4 Detection of PIA and quantification of eDNA

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ACCEPTED MANUSCRIPT Purification and quantification of eDNA release from S. aureus biofilm was conducted as

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previously described [17]. The concentration and purity of the purified DNA were determined

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spectrophotometrically by the absorbance ratio of A260/A280 using a NanoDrop 2000 (Thermo

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Scientific, Waltham, USA) after treatment with TTO for 24 h.

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PIA was measured as previously reported [18]. Briefly, S. aureus strains were grown under static

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conditions at 37 ℃ for 24 h. Cells were then resuspended with 0.5 M EDTA, and proteinase K was then

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added. Serial dilutions of the PIA extract were transferred to a nitrocellulose membrane (Millipore,

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Billerica, USA). The membrane was blocked with 3% (w/v) BSA and subsequently stained with wheat

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germ agglutinin coupled to horseradish peroxidase (WGA–HRP conjugate; Sigma, Saint Louis, USA).

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The HRP activity was detected by an ECL Plus kit (Beyotime, Shanghai, China). The same

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experiments were also performed at 10 and 48 h (data not shown).

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2.5 Growth curves

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Growth curves of S. aureus strain ATCC 29213 were generated. Briefly, an isolated S. aureus

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colony was cultivated in TSB containing 0.25 % glucose (TSB-g) and incubated at 37 ℃. The biofilm

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cell growth was spectrophotometrically measured by a XTT assay at 540 nm at regular time intervals.

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2.6 RNA-seq

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S. aureus biofilm cells were exposed to TTO for 60 min at a concentration of 1/2×MBIC (1

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mg/ml), and 2 samples and 2 control samples were analyzed. Total RNA was extracted from mature

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biofilms and planktonic cells using the RNeasy Mini Kit following the manufacturer’s protocol

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(Cat#74106, Qiagen, German). Total RNA was purified with RNase-free DNase I (Thermo Scientific,

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Waltham, USA). RNA quality was analyzed using a 2100 Bioanalyzer (Agilent Technologies, Santa

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Clara, USA), and RNA was then transformed into TruSeq libraries for sequencing on the Illumina

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ACCEPTED MANUSCRIPT HiSeqX Ten 2*150 bp platform. Briefly, cDNA was synthesized and then subjected to blunt-ending,

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phosphorylation and addition of a single 3’adenosine moiety and Illumina adapters on the repaired ends.

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The cDNA cluster was acquired via a cBot User Guide. The cDNA libraries were generated according

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to the Illumina HiSeqX Ten mRNA-sequencing sample preparation protocol. The sequencing process

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was controlled by means of Illumina data collection software. Total RNA samples were stored at

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−70 °C. A total of 5 µg of each RNA sample was sent to Shanghai Biotechnology Corporation

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(Shanghai, China) for RNA sequencing.

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The resulting high-quality reads were mapped onto the S. aureus strain ATCC 29213 reference

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genome (accession no. NC_002745.2 and NC_003140.1) using Bowtie2 (version: 2-2.0.5). The

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Bowtie2 alignments were processed via BED Tools (version: 2.16.1). The assemblies of mapped reads

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and the calculations of expression values of predicted S. aureus transcripts were represented as

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fragments per kilobase of transcript per million fragments (FPKM) [19]. Significant differences in

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genes were identified with the threshold false discovery rate (FDR) ≤ 0.05 and |fold-change| > 2. These

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samples were analyzed via target prediction, including Gene Ontology (GO) enrichment analysis and

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Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis for DEGs. The same formula

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was used for the pathway enrichment analysis with GO and KEGG. The enriched P values of GO and

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KEGG were calculated according to the hypergeometric test [20]. The parameters for this equation are

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as follows: N is the number of all genes with a GO/KO annotation; n is the number of DEGs in N; M

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represents the number of all genes that have been annotated to specific pathways; and m represents the

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number of DEGs in M. For both GO and KEGG enrichment analyses, we selected a corrected P value

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＜ 0.05 as a threshold to determine significant enrichment of the gene sets. Both GO and KEGG of the

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transcripts were identified trough homemade perl scripts. All the processes discussed above were

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conducted at Shanghai Biotechnology Corporation.

 m  N − M     i  n − i   P = 1− ∑ N i =0   n

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2.7 RT-qPCR

The effect of TTO on expression levels of RNA was examined using oligo dT-primers and

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Superscript II RNAse-Reverse Transcriptase (Takara) to confirm the RNA-seq result. Target gene

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mRNA expression was quantified using SYBR® Premix Ex Taq II (Takara-Bio, Shiga, Japan). The

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primer pairs used in quantitative RT-PCR are listed in Table 1. 16S rRNA was used as the mRNA

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control. Reactions were performed using an ABI Prism 7000 sequence detection system (Applied

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Biosystems, Nieuwerkerk a/d IJssel, The Netherlands) with the following program: 95 ℃ for 15 s, 60 ℃

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for 30 s, and 95 ℃ for 15 s.

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2.8 Statistical analysis

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The statistical analysis of all of the experiments in our study except the RNA-seq were done as

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following: Experimental data were analyzed with SPSS software and compared using Student's t-test.

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The results from quantitative analyses are expressed as the mean ± SD of the data from different

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independent experiments. Differences with a P value of < 0.05 were considered statistically significant.

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All experiments were repeated in triplicate independently.

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3. Results

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3.1 Phenotype analysis of S. aureus biofilm cells with TTO treatment

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The MIC and MBC values for TTO were 1-2 mg/ml (~0.11%-0.22%) and 4-8 mg/ml

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ACCEPTED MANUSCRIPT (~0.44%-0.88%), respectively (Table 2). In the biofilms, the MBIC and MBBC values for TTO

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treatment were 2-4 mg/ml (~0.22%-0.44%) and 32-64 mg/ml (~3.52%-7.05%), respectively (Table 2).

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The MBBC value was 16-32 times the MBIC value, indicating that the biofilm existed enhanced the

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resistance to S. aureus. S. aureus ATCC 29213 was selected for the following experiments in this

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section.

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The inhibition of S. aureus formation by TTO-treated biofilms was tested by the crystal violet

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staining method. As shown in Fig. 1a, higher concentrations of TTO resulted in lower biofilm masses.

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Fig. 1B shows that the TTO-treated (2 mg/ml) biofilm biomass was reduced to 25% of the control.

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The viability of the preformed S. aureus biofilms exposed to TTO (1 to 4 mg/ml) was further

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verified by CLSM analysis. The pre-established biofilms were stained with a LIVE/DEAD BacLight

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bacterial viability kit and imaged using confocal laser scanning microscopy (Fig. 2). Green

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fluorescence indicated SYTO-9 staining (live cells), and red fluorescence indicated propidium iodide

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staining (dead cells). In the control sample, the green fluorescence was strong (Fig. 2A). Treatment of

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biofilm with 1 mg/ml TTO for 48 h decreased the number of bacteria in the biofilm, which increased

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the spaces in the biofilm (Fig. 2B). Exposure to 2 mg/ml TTO resulted in more red fluorescence than

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the 1 mg/ml TTO sample, and treatment with 4 mg/ml TTO increased the red fluorescence even further,

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indicating the most detrimental effects on S. aureus biofilms (Fig. 2C, D). As expected, S. aureus cells

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exposed to 4 mg/ml disrupted the biofilm and induced cell death (Fig. 2D).

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The amount of eDNA in the cell-free supernatants of the biofilms was measured using a

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spectrophotometer and was reported as the eDNA per relative biomass to account for the amount of

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bacteria was present in the biofilms. The average production of eDNA resulting from biofilm exposure

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to 0.5, 1, 2 and 4 mg/ml TTO decreased to approximately 70%, 60%, 40% and 30%, respectively, of

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ACCEPTED MANUSCRIPT the control (Fig. 3A). Levels of PIA production from biofilms exposed to TTO at

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different concentrations are shown in Fig. 3B. The inhibition ratio of PIA expression following

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exposure to 0, 0.5, 1, 2 and 4 mg/ml TTO was 30%, 40%, 60% and 80%, respectively, compared to the

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control. The results showed that there was a significant decrease in eDNA release and PIA production

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in biofilms treated with TTO compared to the control sample. Thus, the observed reduction in eDNA

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and PIA production may be the leading factors responsible for decreased biofilm formation.

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To select the optimal TTO concentration to protect against the S. aureus strain for the

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transcriptional analysis, we generated growth curves for S. aureus ATCC 29213 treated with TTO. The

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growth of S. aureus ATCC 29213 in biofilms as measured by the XTT assay is shown in Fig. 4. A

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steady decrease in the optical density of cells treated with TTO at sub-MBBC concentrations (1, 2, 4

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and 8 mg/ml) was observed versus the control group. The results showed that a strong bacteriostatic

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action against S. aureus biofilm cells was generated by TTO at low concentrations.

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3.2 DEGs in biofilm cells treated with TTO

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To measure the effects of low TTO concentrations on the S. aureus ATCC 29213 strain, we used

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RNA-seq to test the transcriptional profile of biofilm cells at a sub-MBIC concentration (1/2×MBIC, 1

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mg/ml) of TTO at the 60 min time point. Total RNA was extracted from each group, transformed into

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cDNA libraries via Illumina TruSeq Stranded mRNA LT and sequenced using an Illumina HiSeqX Ten

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platform. The raw reads were filtered by Seqtk software for quality control before mapping to genome

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by Bowtie2 (version: 2-2.0.5) [21]. HTSeq was employed to count the fragments of genes followed by

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TMM (trimmed mean of M values) normalization [22]. DEGs were identified as the genes with an

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FDR ≤ 0.05 and |fold-change| > 2 [23]. After data processing, DEGs with P < 0.05 (after correction for

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multiple tests) were listed. A total 304 genes were differentially expressed in biofilm cells treated with

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ACCEPTED MANUSCRIPT TTO versus the control group (without TTO treatment) with 104 genes downregulated (≤ -2-fold) and

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200 genes upregulated (≥ 2-fold) in the biofilm cells (Fig. 5). A complete list of all DEGs from S.

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aureus ATCC 29213 biofilm cells treated with TTO is shown in Table S1 in the Supplementary

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Material. The RNA-seq data were submitted to Gene Expression Omnibus (GEO) under accession

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number GSE85787.

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3.3 Validation of RNA-seq expression by real-time RT-PCR

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To validate the RNA-seq-based transcriptome results, the expression levels of 9 selected genes

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were examined using real time RT-PCR (Table 3). The expression levels of sarA, icaR and cidA were

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downregulated, while the expression levels of sspA, lrgA, fnbB, lytM, glpT and msmX were upregulated.

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Therefore, the results showed that there was no significant difference between real-time RT-PCR and

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RNA-seq data.

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3.4 GO enrichment and KEGG pathway analysis

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GO analysis is considered as an internationally standardized system for classifying gene function, and

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it provides strictly defined concepts and controlled vocabulary to reveal the gene properties and gene

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products in any organism [24]. According to the GO classification analysis (Table S2), the 205 DEGs in

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the TTO-treated biofilm cells versus untreated biofilm cells were classified into three groups as follows:

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molecular functions, cellular components, and biological processes (Fig. 6). We filtered the genes that

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were significantly enriched with a P value < 0.05, and the number of significant terms in the GO

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enrichment was 119.

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Genes generally cooperate with each other to produce biological function. KEGG pathway

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analysis was used to provide insights into the biological functions of genes. We found 107 DEGs in

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KEGG pathways (Table S3) between biofilm cells treated with TTO and untreated biofilm cells.

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ACCEPTED MANUSCRIPT Pathways with a P value <0.05 were considered to be significantly enriched and DEGs. Eighteen

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pathways were significantly enriched in the KEGG analysis, including the phosphotransferase system

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(PTS); valine, leucine and isoleucine biosynthesis; and butanoate metabolism (Fig. 7). We discuss

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several of these important pathway genes involved in biofilms in the discussion.

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Discussion

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TTO has been used to inhibit bacteria in many previous studies [25, 26]. The antimicrobial activity

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of TTO may be due to terpinen-4-ol, which is the main constituent in TTO [27, 28]. However,

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ߛ-terpinene and ߙ-terpinen, other minor compounds present in TTO, may also inhibit antibacterial

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activity by producing a synergistic effect among other components [29]. To date, several studies have

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investigated the effects of TTO on S. aureus biofilms: For example, Brady et al. [30] suggested that

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TTO was effective in eradicating biofilm-grown MRSA at a concentration of 5 % which was similar to

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our study with the MBBC values ranging from 32 mg/ml to 64 mg/ml (~3.52%-7.05%); Kwieciński et

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al. [31] showed 1% TTO destroyed the biofilm formed by S. aureus 8325-4 and Budzyńska et al. [32]

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demonstrated that the MBEC (minimal biofilm eradication concentration) of TTO against S. aureus

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biofilms was 0.78%, which were both a little lower than our study, the differences may due to the

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different contents and components of TTO.

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Cuaron et al. [33] performed a transcriptional profiling experiment (DNA microarrays) with S.

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aureus (planktonic cells) exposed to a growth inhibitory concentration of TTO, and revealed that TTO

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challenge led to the down-regulation of genes involved with energy-intensive transcription and

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translation, and altered the regulation of genes involved with heat shock and cell wall metabolism. In

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our study, we used RNA-Seq for transcriptome analysis instead of DNA microarrays. Comparing to

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microarrays, RNA-Seq is very powerful and cost-efficient for transcriptome analysis [34], and it has

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ACCEPTED MANUSCRIPT replaced microarrays as the preferred technique for gene expression analysis. In order to avoid the

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strain-specific differences to the most extent, we chose S. aureus ATCC 29213 as the standard strain of

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S. aureus for the experiments. Because that the standard strain was used widely and more stable than

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the clinical strains from different countries. The repetition of experiment is good and strain variability

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was lower. In this paper, except the glycine, serine and threonine metabolism pathways, purine

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metabolism pathway and pyrimidine metabolism pathway, we also discussed the differently expressed

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genes associated with biofilm contribution.

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The biofilm matrix is a complex mixture of macromolecules that includes exopolysaccharides,

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proteins and DNA. Our findings suggested that TTO effectively inhibited the release of eDNA and the

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production of PIA during biofilm formation with increasing concentrations of the drug. In addition, the

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results of crystal violet staining and CLSM showed that TTO in different concentrations could

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differently destruct the existing biofilms and inhibit the continuing formation of biofilm. Then, we

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inferred that TTO could could influence S. aureus biofilm formation by affecting eDNA release and

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PIA expression. Therefore, DEGs associated with biofilms were analyzed. The sar locus encodes the

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DNA-binding protein, SarA, which is known to control the production of some matrix adhesion genes,

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including fnbA (encoding fibronectin binding protein A) and icaRA (encoding coagulase) [35-37]. sarA

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has a global effect on many S. aureus virulence genes that seem to play a role in biofilm formation. In

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our study, sarA was downregulated by 1.3-fold. A previous study reported that the expression of sarA is

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decreased by essential oil extracted from C. obtusa leaves, which results in decreased production of

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virulence factors in S. aureus [38]. These results were in accordance with our results, suggesting that

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the inhibition mechanism of TTO on S. aureus may be the same as that for the essential oil extracted

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from C. obtusa leaves. eDNA, which is released in cell autolysis, is an essential matrix molecule in S.

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ACCEPTED MANUSCRIPT aureus biofilm [39]. The release of DNA is regulated by cid and lrg operons [17, 40]. The cidA gene

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encodes a murein hydrolase regulator that induces cell lysis in the course of biofilm formation, whereas

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the lrg operon inhibits cell lysis [41]. In this study, the transcript levels of the positive regulator, cidA,

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were decreased by 1.5-fold, and the transcript levels of the negative regulator of autolysis, lrgA and

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lrgB, were markedly increased by 2.9-fold and 3.9-fold, respectively. Although sarA and cidA were

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downregulated less than 2-fold, the results were validated by the results of real-time RT-PCR, so the

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results were convinced. We found low concentration of TTO increased the expression of icaA, icaB,

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icaC and icaD by 7.5, 5.8, 3.9 and 6.8-fold, respectively. The production of the icaADBC operon

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encoded PIA by S. aureus is one of the most studied mechanisms of biofilm formation, making the ica

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genes a potential target for biofilm inhibitors [42]. Similar effects were observed by Nuryastuti et al.

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[43] and Pimentel-Filho Nde et al. [44] when evaluating subinhibitory concentrations of cinnamon oil

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on the expression of icaA in S. epidermidis and subinhibitory concentrations of bovicin HC5 and nisin

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on the expression of icaD in S. aureus, respectively. They found that even reducing biofilm formation,

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cinnamon oil enhanced icaA expression and bovicin HC5, nisin enhanced icaD expression. The

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different changes of biofilm related genes can provide some evidence on the inhibitory effect of TTO

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on the biofilm formation of S. aureus, especially on the reduction of TTO in eDNA and PIA production.

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A comparison of the complete glycine, serine and threonine metabolism pathways between

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TTO-treated biofilm cells and untreated biofilm cells was performed. The RNA-seq results indicated

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that 12 genes were differentially expressed. Eleven of these genes (sdrE, SA1003, hlgA, pgm, SA1812,

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SA1271, sak, SA1813, asd, SA1016, and sbi) were upregulated by more than 2-fold in the biofilm cells

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treated with TTO compared to the untreated biofilm cells, and 1 of the genes (gpmA) was

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downregulated. Most of the adhesions secreted by S. aureus are cell wall-anchored proteins and are

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ACCEPTED MANUSCRIPT classified into a single family known as members of microbial surface components recognizing

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adhesive matrix molecules [45]. The Sdr proteins are encoded by the tandemly arrayed sdrC, sdrD, and

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sdrE, and they are components of microbial surface-recognizing adhesive matrix molecules. The Sdr

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proteins also have different effects on S. aureus pathogenicity [46]. The allocation of sdrE has been

309

reported to be general among clinical isolates, and it has been suggested that it likely plays a crucial

310

role during infection [47]. The Sak gene encoding staphylokinase (Sak) is a virulence-related gene that

311

may regulate bacterial invasion into host tissues and may improve bacterial resistance to phagocytosis.

312

Interestingly, Kwiecinski et al. [48] reported that plasmin(ogen)-dependent proteolysis and fibrinolysis

313

can be accelerated by Sak, which in turn prevents bacterial adhesion to surfaces and destroys biofilm

314

matrices, resulting in the collapse of biofilm architecture and bacterial separation. This phenomenon

315

may explain our results. Thus, we can infer that the production of plasmin(ogen)-dependent proteolysis

316

and fibrinolysis in the group treated with TTO was more than that of the untreated group. GpmA, which

317

was down-regulated in this study, may activate the interconversion of 2-phosphoglycerate and

318

3-phosphoglycerate. As a vital enzyme in glycolysis and energy metabolism, GpmA is regarded as a

319

underlying target for novel antibiotics [49].

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The biofilm cells treated with TTO had 10 genes involved in purine metabolism pathway that

321

were differentially expressed. Five (purQ, purC, purL, SA1172, and purK) of these genes were

322

upregulated, and 5 (holB, ureC, nurD, SA0515, and nrdF) of these genes were downregulated.

323

Brugarolas et al. [50] reported that purK is a bacterial enzyme that is an attractive antibacterial drug

324

target. The pathogenesis of staphylococcal infections is multifactorial, and golden pigment is a

325

representative feature of staphylococcal infections. Golden pigment can protect the S. aureus from

326

oxidation-based clearance. Lan et al. stated that S. aureus production of golden pigment can be

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ACCEPTED MANUSCRIPT influenced by metabolic pathways, such as purine biosynthesis. Lan et al. [51] also reported that

328

deactivation of purine biosynthetic genes (purN, purH, purD or purA) may account for the intensive

329

pigmentation. Thus, we can speculate that the upregulation of purQ, purC, purL, and purK in the

330

biofilm cells after treatment with TTO led to attenuated golden pigment. The aerobic class Ib encoded

331

by the S. aureus class Ib RNR nrdIEF is an oxygen-dependent enzyme. The anaerobic class III

332

ribonucleotide reductase encoded by class III RNR nrdDG genes is essential for anaerobic growth. The

333

transcription of class III nrdDG genes is at least 10-fold higher under anaerobic than under aerobic

334

conditions. In contrast, there is no important effect of oxygen concentration on the transcription of class

335

Ib nrdIEF genes[52]. Wu et al. [53] reported that the ∆srrA mutant strain produces less PIA and shows

336

less initial adherence capacity, and their microarray analysis revealed that the srrA mutation influences

337

transcription of 230 genes in the microaerobic case and 51 genes in the oxic case (including nrdDG).

338

Therefore, we inferred that the downregulation of nrdD and nrdF was related to the mechanism of S.

339

aureus biofilm formation.

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The biofilm cells treated with TTO resulted in 8 genes involved in the pyrimidine metabolism

341

pathway to be differentially expressed in biofilms. Five (pyrB, pyrAA, pyrF, pyrC, and pryAB) of these

342

genes were upregulated, and 3 (holB, nrdD, and nrdF) of these genes were downregulated.

343

Uridine-5'-triphosphate disodium salt (UMP), known as the precursor of other pyrimidine nucleotides

344

is encoded by pyrE. Jensen et al. [54] reported that mutations can result in high constitutive expression

345

of pyrE (and of pyrB) under the condition of high cellular uridine monophosphate (UTP)

346

concentrations. The expression of pyrE, pyrB and pyrF is mediated primarily by intracellular UTP and

347

is high when the UTP pool is at a low concentration [55]. However, the pyrC gene encoding

348

dihydroorotase is negatively regulated by CTP and is stimulated by GTP [56]. Dihydroorotase encoded

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16

ACCEPTED MANUSCRIPT by pyrC is the third enzyme in the bacterial pyrimidine biosynthesis pathway of S. aureus. PyrC has

350

been demonstrated to be essential in S. aureus and is an underlying target for the development of

351

selective antibacterial agents against S. aureus [57]. In the presence of ciprofloxacin, erythromycin and

352

vancomycin, both purine and pyrimidine metabolic pathways are the important affected pathways [58].

353

TTO showed good activity against S. aureus in both biofilm and planktonic cells, and it

354

significantly affected the expression of several genes related to S. aureus biofilm formation. These

355

findings may provide insight into understanding the response mechanisms of S. aureus to TTO, and

356

these results promote additional research on TTO as an antibacterial compound.

357

Acknowledgments

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The study was supported by grants from the National Nature Science Foundation of China (No.

359

31271951 and No. 81573448), China Postdoctoral Science Foundation (2013M530142), the Program

360

for New Century Excellent Talents in University (NCET-13-024) and Natural Science Foundation of

361

Jilin Province (20150101009JC).

362

References

363

[1] Ghosh A, Ricke SC, Almeida G, Gibson KE. Combined Application of Essential Oil Compounds

364

and Bacteriophage to Inhibit Growth of Staphylococcus aureus In Vitro. Curr Microbiol.

366 367

EP

AC C

365

TE D

358

2016;72:426-35.

[2] Bazargani MM, Rohloff J. Antibiofilm activity of essential oils and plant extracts against Staphylococcus aureus and Escherichia coli biofilms. Food Control. 2016;61:156-64.

368

[3] Teixeira PC, Leite GM, Domingues RJ, Silva J, Gibbs PA, Ferreira JP. Antimicrobial effects of a

369

microemulsion and a nanoemulsion on enteric and other pathogens and biofilms. Int J Food

370

Microbiol. 2007;118:15-9.

17

ACCEPTED MANUSCRIPT 371

[4] Lindsay D, von Holy A. Bacterial biofilms within the clinical setting: what healthcare professionals should know. J Hosp Infect. 2006;64:313-25.

372

[5] Ceri H, Olson ME, Stremick C, Read RR, Morck D, Buret A. The Calgary Biofilm Device: new

374

technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. J Clin

375

Microbiol. 1999;37:1771-6.

RI PT

373

376

[6] Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. Lancet. 2001;358:135-8.

377

[7]

I,

Vinogradov

E,

Flahaut

S,

Kogan

G,

Jabbouri

SC

Sadovskaya

S.

Extracellular

carbohydrate-containing polymers of a model biofilm-producing strain, Staphylococcus

379

epidermidis RP62A. Infect Immun. 2005;73:3007-17.

380 381

M AN U

378

[8] Nazzaro F, Fratianni F, De Martino L, Coppola R, De Feo V. Effect of essential oils on pathogenic bacteria. Pharmaceuticals (Basel). 2013;6:1451-74.

[9] Cross SE, Russell M, Southwell I, Roberts MS. Human skin penetration of the major components

383

of Australian tea tree oil applied in its pure form and as a 20% solution in vitro. European Journal

384

of Pharmaceutics and Biopharmaceutics. 2008;69:214-22.

387 388 389 390 391 392

EP

386

[10] Hammer KA, Carson CF, Riley TV. Antifungal activity of the components of Melaleuca alternifolia (tea tree) oil. J Appl Microbiol. 2003;95:853-60.

AC C

385

TE D

382

[11] Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nature Reviews Genetics. 2009;10:57-63.

[12] Williams CR, Baccarella A, Parrish JZ, Kim CC. Trimming of sequence reads alters RNA-Seq gene expression estimates. BMC Bioinformatics. 2016;17. [13] Ozsolak F, Platt AR, Jones DR, Reifenberger JG, Sass LE, McInerney P, et al. Direct RNA sequencing. Nature. 2009;461:814-U73.

18

ACCEPTED MANUSCRIPT 393 394

[14] CLSI. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically (8th ed., pp. M7eA8). Wayne, PA, USA: CLSI Approved Standard. 2009. [15] Xing M, Shen, F, Liu L, Chen Z, Guo N, Wang X, Wang W, Zhang K, Wu X, Wang X, Li Y, Sun S,

396

Yu L. Antimicrobial efficacy of the alkaloid harmaline alone and in combination with

397

chlorhexidine digluconate against clinical isolates of Staphylococcus aureus grown in planktonic

398

and biofilm cultures. Lett Appl Microbiol. 2012; 54: 475–482.

RI PT

395

[16] Guo N, Zhao X, Li W, Shi C, Meng R, Liu Z, et al. The synergy of berberine chloride and totarol

400

against Staphylococcus aureus grown in planktonic and biofilm cultures. J Med Microbiol.

401

2015;64:891-900.

M AN U

SC

399

[17] Rice KC, Mann EE, Endres JL, Weiss EC, Cassat JE, Smeltzer MS, et al. The cidA murein

403

hydrolase regulator contributes to DNA release and biofilm development in Staphylococcus

404

aureus. Proc Natl Acad Sci U S A. 2007;104:8113-8.

TE D

402

[18] Lou Q, Zhu T, Hu J, Ben H, Yang J, Yu F, et al. Role of the SaeRS two-component regulatory

406

system in Staphylococcus epidermidis autolysis and biofilm formation. BMC Microbiol.

407

2011;11:146.

409 410 411 412 413 414

[19] Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, et al. Transcript

AC C

408

EP

405

assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010;28:511-U174.

[20] Zhang B, Kirov S, Snoddy J. WebGestalt: an integrated system for exploring gene sets in various biological contexts. Nucleic Acids Res. 2005;33:W741-W8. [21] Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nature methods. 2012; 9: 357-359.

19

ACCEPTED MANUSCRIPT

416 417 418

[22] Anders S, Pyl PT, Huber W. 2015. HTSeq--a Python framework to work with high-throughput sequencing data. Bioinformatics. 2015; 31: 166-169. [23] Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010; 26: 139-140.

RI PT

415

[24] Xiu L, Fu YB, Deng Y, Shi XJ, Bian ZY, Ruhan A, et al. Deep sequencing-based analysis of gene

420

expression in bovine mammary epithelial cells after Staphylococcus aureus, Escherichia coli, and

421

Klebsiella pneumoniae infection. Gen Mol Res. 2015;14:16948-65.

SC

419

[25] Goni MG, Tomadoni B, Roura SI, Moreira MR. Effect of Preharvest Application of Chitosan and

423

Tea Tree Essential Oil on Postharvest Evolution of Lettuce Native Microflora and Exogenous

424

Escherichia Coli O157: H7. J Food Saf. 2014;34:353-60.

M AN U

422

[26] Mertas A, Garbusinska A, Szliszka E, Jureczko A, Kowalska M, Krol W. The influence of tea tree

426

oil (Melaleuca alternifolia) on fluconazole activity against fluconazole-resistant Candida albicans

427

strains. Biomed Res Int. 2015;2015:590470.

430 431 432 433

and other medicinal properties. Clin Microbiol Rev. 2006;19:50-62.

EP

429

[27] Carson CF, Hammer KA, Riley TV. Melaleuca alternifolia (tea tree) oil: a review of antimicrobial

[28] Cox SD, Mann CM, Markham JL. Interactions between components of the essential oil of

AC C

428

TE D

425

Melaleuca alternifolia. J Appl Microbiol. 2001;91:492-7.

[29] Marino M, Bersani C, Comi G. Impedance measurements to study the antimicrobial activity of essential oils from Lamiaceae and Compositae. Int J Food Microbiol. 2001;67:187-95.

434

[30] Brady A, Loughlin R, Gilpin D, Kearney P, Tunney M. In vitro activity of tea-tree oil against

435

clinical skin isolates of meticillin-resistant and -sensitive Staphylococcus aureus and

436

coagulase-negative staphylococci growing planktonically and as biofilms. J Med Microbiol. 2006;

20

ACCEPTED MANUSCRIPT 437

55: 1375-80. [31] Kwieciński J, Eick S, Wójcik K. Effects of tea tree (Melaleuca alternifolia) oil on Staphylococcus

439

aureus in biofilms and stationary growth phase. Int J Antimicrob Agents. 2009; 33(4): 343-7.

440

[32] Budzyńska A, Wieckowska-Szakiel M, Sadowska B, Kalemba D, Rózalska B. Antibiofilm

441

Activity of Selected Plant Essential Oils and their Major Components. Pol J Microbiol. 2011;

442

60(1): 35-41.

RI PT

438

[33] Cuaron JA, Dulal S, Song Y, Singh AK, Montelongo CE, Yu W, Nagarajan V, Jayaswal RK,

444

Wilkinson BJ, Gustafson JE. Tea Tree Oil-Induced Transcriptional Alterations in Staphylococcus

445

aureus. Phytother Res. 2013; 27(3): 390-396.

447

M AN U

446

SC

443

[34] Williams CR, Baccarella A, Parrish JZ, Kim CC. Trimming of sequence reads alters RNA-Seq gene expression estimates. BMC Bioinformatics. 2016; 17: 103.

[35] Arya R, Ravikumar R, Santhosh RS, Princy SA. SarA based novel therapeutic candidate against

449

Staphylococcus aureus associated with vascular graft infections. Front Microbiol. 2015;6:416.

450

[36] Fuda CCS, Fisher JF, Mobashery S. beta-Lactam resistance in Staphylococcus aureus: the adaptive

453 454

EP

452

resistance of a plastic genome. Cell Mol Life Sci. 2005;62:2617-33. [37] Kupferwasser LI, Yeaman MR, Nast CC, Kupferwasser D, Xiong YQ, Palma M, et al. Salicylic

AC C

451

TE D

448

acid attenuates virulence in endovascular infections by targeting global regulatory pathways in Staphylococcus aureus. J Clin Invest. 2003;112:222-33.

455

[38] Kim ES, Kang SY, Kim YH, Lee YE, Choi NY, You YO, et al. Chamaecyparis obtusa Essential Oil

456

Inhibits Methicillin-Resistant Staphylococcus aureus Biofilm Formation and Expression of

457

Virulence Factors. J Med Food. 2015;18:810-7.

458

[39] Otto M. Staphylococcal Infections: Mechanisms of Biofilm Maturation and Detachment as Critical

21

ACCEPTED MANUSCRIPT 459

Determinants of Pathogenicity. Annual Review of Medicine, Vol 64. 2013;64:175-88. [40] Shen F, Tang X, Wang Y, Yang Z, Shi X, Wang C, et al. Phenotype and expression profile analysis

461

of Staphylococcus aureus biofilms and planktonic cells in response to licochalcone A. Appl

462

Microbiol Biotechnol. 2015;99:359-73.

463 464

RI PT

460

[41] Sadykov MR, Bayles KW. The control of death and lysis in staphylococcal biofilms: a coordination of physiological signals. Curr Opin Microbiol. 2012;15:211-5.

[42] Oduwole KO, Glynn AA, Molony DC, Murray D, Rowe S, Holland LM, McCormack DJ, O'Gara

466

JP. Anti-biofilm activity of sub-inhibitory povidone-iodine concentrations against Staphylococcus

467

epidermidis and Staphylococcus aureus. J Orthop Res. 2010; 28, 1252-1256.

M AN U

SC

465

[43] Nuryastuti T, van derMei HC, Busscher HJ, Iravati S, Aman AT, Krom BP. Effect of cinnamon oil

469

on icaA expression and biofilm formation by Staphylococcus epidermidis. Appl Environ Microbiol.

470

2009; 75: 6850-6855.

TE D

468

[44] Pimentel-Filho Nde J, Martins MC, Nogueira GB, Mantovani HC, Vanetti MC. Bovicin HC5 and

472

nisin reduce Staphylococcus aureus adhesion to polystyrene and change the hydrophobicity profile

473

and Gibbs free energy of adhesion. Int J Food Microbiol. 2014; 190: 1-8.

475 476

[45] Liu HL, Lv JN, Qi XQ, Ding Y, Li D, Hu LH, et al. The carriage of the serine-aspartate repeat

AC C

474

EP

471

protein-encoding sdr genes among Staphylococcus aureus lineages. Brazilian Journal of Infectious Diseases. 2015;19:498-502.

477

[46] Josefsson E, McCrea KW, Ni Eidhin D, O'Connell D, Cox J, Hook M, et al. Three new members

478

of the serine-aspartate repeat protein multigene family of Staphylococcus aureus. Microbiology.

479

1998;144 ( Pt 12):3387-95.

480

[47] Hair PS, Foley CK, Krishna NK, Nyalwidhe JO, Geoghegan JA, Foster TJ, et al. Complement

22

ACCEPTED MANUSCRIPT 481

regulator C4BP binds to Staphylococcus aureus surface proteins SdrE and Bbp inhibiting bacterial

482

opsonization and killing. Results Immunol. 2013;3:114-21. [48] Kwiecinski J, Peetermans M, Liesenborghs L, Na ML, Bjornsdottir H, Zhu XF, et al.

484

Staphylokinase Control of Staphylococcus aureus Biofilm Formation and Detachment Through

485

Host Plasminogen Activation. J Infect Dis. 2016;213:139-48.

RI PT

483

[49] Davies DR, Staker BL, Abendroth JA, Edwards TE, Hartley R, Leonard J, et al. An ensemble of

487

structures of Burkholderia pseudomallei 2,3-bisphosphoglycerate-dependent phosphoglycerate

488

mutase. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011;67:1044-50.

M AN U

SC

486

[50] Brugarolas P, Duguid EM, Zhang W, Poor CB, He C. Structural and biochemical characterization

490

of N5-carboxyaminoimidazole ribonucleotide synthetase and N5-carboxyaminoimidazole

491

ribonucleotide mutase from Staphylococcus aureus. Acta Crystallogr D Biol Crystallogr.

492

2011;67:707-15.

493 494

TE D

489

[51] Lan L, Cheng A, Dunman PM, Missiakas D, He C. Golden pigment production and virulence gene expression are affected by metabolisms in Staphylococcus aureus. J Bacteriol. 2010;192:3068-77. [52] Masalha M, Borovok I, Schreiber R, Aharonowitz Y, Cohen G. Analysis of transcription of the

496

Staphylococcus aureus aerobic class Ib and anaerobic class III ribonucleotide reductase genes in

AC C

497

EP

495

response to oxygen. J Bacteriol. 2001;183:7260-72.

498

[53] Wu YC, Wu Y, Zhu T, Han HY, Liu HY, Xu T, et al. Staphylococcus epidermidis SrrAB Regulates

499

Bacterial Growth and Biofilm Formation Differently under Oxic and Microaerobic Conditions. J

500

Bacteriol. 2015;197:459-76.

501

[54] Kaj Frank Jensen, Jan Neuhard, Schack L. RNA polymerase involvement in the regulation of

502

expression of Salmonella typhimurium pyr genes. Isolation and characterization of a

23

ACCEPTED MANUSCRIPT 503

fluorouracil-resistant mutant with high, constitutive expression of the pyrB and pyrE genes due to

504

a mutation in rpoBC. The EMBO Joumal. 1982;1:69-74. [55] Peter Poulsen, Fons Bonekamp, Jensen KF. Structure of the Escherichia coli pyrE operon and

506

control of pyrE expression by a UTP modulated intercistronic attentuation. The EMIBO Journal.

507

1984;3:1783-90.

509

[56] Sorensen KI, Neuhard J. Dual transcriptional initiation sites from the pyrC promoter control expression of the gene in Salmonella typhimurium. Mol Gen Genet. 1991;225:249-56.

SC

508

RI PT

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[57] Truong L, Hevener KE, Rice AJ, Patel K, Johnson ME, Lee H. High-level expression, purification,

511

and characterization of Staphylococcus aureus dihydroorotase (PyrC) as a cleavable His-SUMO

512

fusion. Protein Expression Purif. 2013;88:98-106.

[58] Dorries K, Schlueter R, Lalk M. Impact of Antibiotics with Various Target Sites on the Metabolome

515

2014;58:7151-63.

Staphylococcus

aureus.

AC C

516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533

of

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24

Antimicrobial

Agents

and

Chemotherapy.

ACCEPTED MANUSCRIPT

Table 1 Primers used in real-time RT-PCR with SYBR green probes Primer

Sequence (5'-3')

16S rRNA

16S rRNAfor

CGTGCTACAATGGACAATACAAA

16S rRNArev

ATCTACGATTACTAGCGATTCCA

sarA for

TCTTGTTAATGCACAACAACGTAA

sarA rev

TGTTTGCTTCAGTGATTCGTTT

icaR for

CAATAATCTAATACGCCTGAG

icaR rev

AGTAGCGAATACACTTCATCT

sspA for

CGATCGTCACCAAATCACAGA

sspA rev

TGCGTAGCATCTACGACGTGT

cidA for

CTTAGCCGGCAGTATTGTTGC

cidA rev

TGAAGATAATGCAACGATAC

sarA icaR sspA cidA

fnbB

GTGACATAGCCAGTACAAAT

fnbB rev

AACTTGGAAAAATGGCGTTG

lytMfor

ACGGTGTCGACTATGCAATGC

lytMrev

TACTTGATTGCCGCCACCA

glpTfor

CGACTTTGCTACAAGCGATAA

glpTrev

CGCCCAATCAAGTACACCA

msmXfor

CATTTGGGCTAAAGCTACG

msmXrev

GACGCTGTCCACCAGATAA

TE D EP

AC C

538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554

lrgA rev

TTCTGCATGACCTTCTGCA

glpT

537

CTGGTGCTGTTAAGTTAGGCG

fnbB for

lytM

msmX

lrgA for

M AN U

lrgA

RI PT

Target gene

SC

534 535 536

25

ACCEPTED MANUSCRIPT 555

Table 2 Antimicrobial activities of TTO against 19 S. aureus strains growing in planktonic and biofilm. Planktonic

MIC

MBC

(mg/ml)

S.aureus JL-20110

1 (1)

2(2)

S.aureus JL-20111

1 (1)

2(2)

S.aureus JL-20112

1 (1)

2(2-4)

S.aureus JL-20113

2 (1-2)

2(2)

S.aureus JL-20114

1 (1)

2(2)

S.aureus JL-20115

1 (1)

2(2)

S.aureus JL-20116

1 (1)

2(2-4)

S.aureus JL-20117

2 (1-2)

S.aureus JL-20118

MBIC

(mg/ml) MBBC

2(2)

(mg/ml)

64(64) 64(64)

2 (2)

32(32)

4 (4)

64(64)

2 (2-4)

32(32-64)

2 (2)

32(32)

2 (2)

32(32)

4(4)

2 (2-4)

64(32-64)

2 (1-2)

2(2)

2 (2)

64(64)

S.aureus JL-20119

1 (1)

2(2)

2 (2)

32(32)

S.aureus JL-20120

2 (1-2)

2(2-4)

2 (2)

32(32)

S.aureus JL-20121

1 (1)

2(2)

2(2)

32(32-64)

2 (1-2)

2(2)

4 (2-4)

32(32)

1 (1)

2(2)

2 (2)

32(32)

1 (1)

2(2)

4 (4)

32(32)

S.aureus JL-20125

1 (1)

2(2-4)

2 (2)

64(64)

S.aureus JL-20126

1 (1)

2(2)

2 (2)

32(32-64)

S.aureus JL-20127

1 (1)

2(2)

2 (2)

64(64)

S.aureus ATCC 29213

1 (1)

2(2)

2 (2)

32(32)

AC C

EP

S.aureus JL-20124

M AN U

TE D

S.aureus JL-20123

SC

2 (2)

S.aureus JL-20122

556 557 558 559 560 561 562 563

(mg/ml)

RI PT

Strains

Biofilm

Values in the brackets represent the range of MIC (MBC, MBIC and MBBC) for TTO.

26

ACCEPTED MANUSCRIPT 564 565 566

Table 3 Selected S. aureus genes that displayed altered expression after TTO treatment of biofilm as determined by microarray analysis and real-time RT–PCR. Fold change± SD Gene

Description

sarA icaR

RNA-seq

transcriptional regulator

-1.5±0.1

-1.3±0.1

biofilm operon icaADBC HTH-type

-2.8±1.1

-2.0±0.8

negative transcriptional regulator IcaR

glutamyl endopeptidase

+2.7±0.5

+2.5±0.3

cidA

riboflavin biosynthesis protein

-1.8±0.3

-1.5±0.2

lrgA

elastin binding protein

+3.0±0.8

+2.9±0.6

fnbB

fibronectin-binding protein A

+2.9±0.6

+2.5±0.5

lytM

glycyl-glycine endopeptidase LytM

+2.6±0.5

+2.5±0.4

glpT

glycerol-3-phosphate transporter

+5.8±1.5

+5.2±1.2

msmX

sugar ABC transporter ATP-binding protein

+44.0±3.7

+39.0±2.9

EP

TE D

M AN U

SC

sspA

AC C

567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590

RI PT

RT-PCR

27

ACCEPTED MANUSCRIPT 591 592 593 594 (A)

Control

1mg/ml

2mg/ml

4mg/ml

RI PT

595 596 597

SC

598 599 (B)

600

602

2.5 Biofilm(A595)

601

M AN U

3

*

2 1.5 1

**

0

604

TE D

603

0.5

Control

1

2

**

4

605 606

EP

Concentration of of TTO(mg/mL) Concertration TTO (mg/ml)

Fig. 1. Effect of TTO on S. aureus biofilm formation determined by crystal violet staining. Bars

608

indicate the mean A595 values of three independent experiments, and experiments were performed in

609

triplicate wells. Values are expressed as the means ± standard deviations; * P<0.05, ** P<0.01.

610

AC C

607

611 612 613

28

ACCEPTED MANUSCRIPT 614 615

618

AC C

617

EP

TE D

M AN U

SC

RI PT

616

619

Fig. 2. CLSM image of LIVE/DEAD stained S. aureus biofilms grown on coverslips. Green (viable

620

cells) and red (dead cells). (A) Control cells (no treated). (B) Cells treated with TTO (1 mg/ml). (C)

621

Cells treated with TTO (2 mg/ml). (D) Cells treated with TTO (4 mg/ml).

622 623

29

624

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SC

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Fig. 3. (A) Effect of eDNA release on S. aureus ATCC 29213. The amount of eDNA in the cell-free

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supernatants from S. aureus biofilms treated with different concentrations of TTO was measured by

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spectrophotometry after 24 h. The values are expressed as nanograms of eDNA per relative biofilm

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biomass (OD600). (B) Effect of TTO on PIA production by S. aureus ATCC 29213. PIA in cultures

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treated with different concentrations of TTO was extracted from the biofilms after 24 h and detected

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using WAG-HRP. The spot was visualized by chemiluminescence detection. Values are expressed as

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the means ± standard deviations; * P < 0.05, ** P < 0.01.

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ACCEPTED MANUSCRIPT 632 633 634

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635

636

640 641 642 643 644

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Fig. 4. Growth curve for S. aureus ATCC 29213 in biofilms treated with TTO.

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645 646 647

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ACCEPTED MANUSCRIPT 648 649 650

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651 652

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653

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654 655

Fig. 5. The volcano plot of DEGs in biofilm cells treated with TTO versus the control group (without

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TTO treatment). The results showed that 304 genes were differentially expressed with 104

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downregulated genes (≤ -2-fold, FDR ≤ 0.05) and 200 upregulated genes (≥ 2-fold, FDR ≤ 0.05) in

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biofilm cells.

659 660 661

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663 664

Fig. 6. Bar plot of GO enrichment results. The number of significant terms in GO enrichment was 119.

668 669 670 671 672 673 674

EP

667

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675 676 677 678

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ACCEPTED MANUSCRIPT 679 680 681 682

686 687 688 689 690

Fig. 7. Bar plot of KEGG enrichment results. The number of significant pathways in KEGG

EP

685

enrichment was 18.

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683

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ACCEPTED MANUSCRIPT Highlights 1. The inhibitory effect of TTO on S. aureus biofilms was determined 2. 304 genes were differentially expressed treated with TTO versus the control biofilm

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3. GO enrichment and KEGG pathway analysis were provided by RNA-seq