Cigarette Smoke Increases Staphylococcus aureus Biofilm Formation via Oxidative Stress

Ritwij Kulkarni, Swati Antala, Alice Wang, Fbio E. Amaral, Ryan Rampersaud, Samuel J. LaRussa, Paul J. Planet and Adam J. Ratner Infect. Immun. 2012, 80(11):3804. DOI: 10.1128/IAI.00689-12. Published Ahead of Print 13 August 2012. Downloaded from http://iai.asm.org/ on November 8, 2012 by ASM- N Street

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Cigarette Smoke Increases Staphylococcus aureus Biolm Formation via Oxidative Stress
Ritwij Kulkarni, Swati Antala, Alice Wang, Fbio E. Amaral, Ryan Rampersaud, Samuel J. LaRussa, Paul J. Planet, and Adam J. Ratner
Department of Pediatrics, Columbia University, New York, New York, USA

The strong epidemiological association between cigarette smoke (CS) exposure and respiratory tract infections is conventionally attributed to immunosuppressive and irritant effects of CS on human cells. Since pathogenic bacteria such as Staphylococcus aureus are members of the normal microbiota and reside in close proximity to human nasopharyngeal cells, we hypothesized that bioactive components of CS might affect these organisms and potentiate their virulence. Using Staphylococcus aureus as a model organism, we observed that the presence of CS increased both biolm formation and host cell adherence. Analysis of putative molecular pathways revealed that CS exposure decreased expression of the quorum-sensing agr system, which is involved in biolm dispersal, and increased transcription of biolm inducers such as sarA and rbf. CS contains bioactive compounds, including free radicals and reactive oxygen species, and we observed transcriptional induction of bacterial oxidoreductases, including superoxide dismutase, following exposure. Moreover, pretreatment of CS with an antioxidant abrogated CS-mediated enhancement of biolms. Exposure of bacteria to hydrogen peroxide alone increased biolm formation. These observations are consistent with the hypothesis that CS induces staphylococcal biolm formation in an oxidant-dependent manner. CS treatment induced transcription of fnbA (encoding bronectin binding protein A), leading to increased binding of CS-treated staphylococci to immobilized bronectin and increased adherence to human cells. These observations indicate that the bioactive effects of CS may extend to the resident microbiota of the nasopharynx, with implications for the pathogenesis of respiratory infection in CS-exposed humans. taphylococcus aureus is a Gram-positive pathogen that colonizes the skin and mucosal spaces of human hosts (42, 50), with a population-wide carriage rate estimated at 20 to 32% (59, 72). It causes a wide range of community- and hospital-acquired infections ranging in severity from uncomplicated cellulitis to deep-seated infections such as endocarditis and osteomyelitis (10). The emergence and spread of methicillin-resistant S. aureus (MRSA) is a global public health issue (29, 41). Much progress has been made in determining the roles of various staphylococcal virulence factors in the pathogenesis of infection (32). However, the underlying mechanisms of the transition of S. aureus from asymptomatic colonizer to human pathogen remain unclear and may involve the concerted activity of host, bacterial, and possibly environmental factors. Cigarette smoking is another important global health threat. Despite intensive public health interventions, rates of smoking remain high in the United States (11) and worldwide (71). Smoking causes a tremendous health burden, not only in smokers but in those exposed to second-hand cigarette smoke as well (24). The adverse health outcomes associated with cigarette smoke, including carcinogenesis (62), promotion of atherosclerosis (56), and chronic lung disease (74), are generally thought to result from the direct action of components of cigarette smoke (CS) on human cells. CS contains a plethora of bioactive compounds, including oxidant, genotoxic, and immunomodulatory factors (15, 57). CS exposure increases the risk of several infectious diseases (25, 33, 73). We recently demonstrated that CS could impair epithelial innate immune responses to microbial products (37), perhaps setting the stage for overgrowth and invasion. Because the colonizing microbiota inhabits human mucosal spaces, microbes may share exposure to a variety of environmental stimuli, including CS. We hypothesized that exposure of S.

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aureus to CS might induce pathways relevant to both survival and pathogenesis. We focused on adherence and biolm formation, which are important determinants of S. aureus colonization and disease (32). We observed that CS exposure increased staphylococcal biolm formation in a rapid, dose-dependent, and oxidantmediated manner. CS enhanced S. aureus transcription of the bronectin binding protein A gene (fnbA), leading to increased staphylococcal binding to human bronectin and increased adherence to human lung adenocarcinoma cell line A549. These observations emphasize the importance of understanding the effects of environmental stimuli, including CS exposure, on the colonizing microbiota rather than solely on host cells.
MATERIALS AND METHODS
Reagents. Unless otherwise specied, reagents were purchased from Sigma. Bacterial strains, cell lines, and preparation of cigarette smoke extract. Staphylococcus aureus strains RN6390 (54), Newman (49), USA300 (65), and 502A (RN6607, NRS149 spa type 230) were grown in tryptic soy broth (TSB) without antibiotic selection. For assays of biolm formation, dextrose-free TSB supplemented with 0.5% glucose (TSBG) was used. Immortalized human upper airway epithelial cell line A549 (ATCC CCL-

Received 6 July 2012 Returned for modication 26 July 2012 Accepted 4 August 2012 Published ahead of print 13 August 2012 Editor: J. B. Bliska Address correspondence to Adam J. Ratner, ar127@columbia.edu. Supplemental material for this article may be found at http://iai.asm.org/. Copyright 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/IAI.00689-12

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185) was maintained in minimal essential medium (MEM) (Gibco) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate (Cellgro), and 10 g/ml ciprooxacin (Fisher). Fresh cigarette smoke extract (CSE) was prepared for each experiment by bubbling smoke from three Marlboro cigarettes into 20 ml TSBG (100% CSE). In the absence of specic dosage data regarding exposure of colonizing microbes to CS, this maximum dose (3 cigarettes/20 ml) was chosen in order to approximate heavy direct CS exposure and is consistent with doses reported to be used elsewhere in the literature (22, 48). Effects were tested over a range of CS concentrations. Bacterial exposure to CSE. Overnight bacterial cultures in TSB were centrifuged, and pellets were resuspended in TSBG. These were exposed to various concentrations of CSE for biolm formation assay or quantitative real-time PCR (qRT-PCR) analysis as described below. Alternatively, short-term CSE exposure was performed as follows. Overnight S. aureus cultures were diluted 1:40 in TSBG and grown to an optical density at 600 nm (OD600) of 1, cultures were centrifuged, and pellets were exposed to 0 or 100% CSE for 1 h. Following short-term stimulation, bacteria were washed to remove residual CSE and tested for biolm formation, adherence, or gene expression by qRT-PCR as described below. Biolm formation assay. The protocol for biolm formation was published earlier (18). In brief, overnight cultures of bacteria were grown to an OD600 of 0.6 to 1.0 in TSB, washed in TSBG, and resuspended at a 1:40 dilution in TSBG with various concentrations of CSE (0, 10, 25, and 50%) or H2O2 (0, 5, and 10 mM) in 96-well plates. These were incubated at 37C with horizontal rotation (150 rpm) overnight. Planktonic bacteria were discarded, and biolms were washed gently with 0.9% NaCl solution three times. Biolm biomass was then xed by baking the plates at 60C for 1 h, followed by staining with 0.3% crystal violet for 15 min at room temperature. Excess stain was removed with water. Plates were dried, biolm-associated stain extracted in a 70% ethanol10% methanol mixture, and absorbance (590 nm) determined. For enumerating planktonic and sessile CFU, following overnight incubation as described above, supernatants were collected gently without disturbing the biolms, following which biolm-associated biomass was scraped off the bottoms of the wells. Serial dilutions of the supernatant and biolm fractions were plated on tryptic soy agar and colonies counted after overnight incubation at 37C. qRT-PCR. Total RNA was extracted from bacteria grown in the presence or absence of CSE for the indicated times using the Ribopure bacterial RNA extraction kit (Ambion) according to the manufacturers instructions. RNA was reverse transcribed to cDNA using the high-capacity cDNA reverse transcription kit (Applied Biosystems.) Quantitative realtime PCR (qRT-PCR) was carried out using Power SYBR green master mix in a StepOne Plus thermal cycler (Applied Biosystems). The list of primers used for qRT-PCR is shown in Table 1. Relative quantication (RQ) values were calculated using a comparative threshold cycle (CT) program on the StepOne software version 2.0. Fibronectin binding assay. Human bronectin, reconstituted as a 2.5-mg/ml stock in Dulbeccos phosphate-buffered saline (D-PBS) and diluted to specied concentrations (20, 10, 5, 2.5, and 1.25 g/ml), was used to coat 96-well at-bottom enzyme-linked immunosorbent assay (ELISA) plates overnight at 4C. S. aureus was exposed to 100% CSE or TSBG alone using the short-term exposure protocol described above, washed in sterile D-PBS twice, and resuspended in sterile D-PBS (the nal OD600 was adjusted to 0.450.) Fibronectin-coated ELISA plates were washed with washing buffer (0.05% Tween 20 in PBS) and blocked for 1 h at 37C using 100 l blocking buffer (washing buffer plus 1% bovine serum albumin [BSA]). Plates were washed again and 100 l of bacteria added to appropriate wells. Following incubation at 37C for 2 h, plates were washed to remove nonadherent bacteria and dried. Adherent bacteria (AB) were xed by incubating with 25% formaldehyde solution for 30 min. Following an additional cycle of washing and drying, adherent bacteria were quantied using the crystal violet assay as described above.

TABLE 1 Primers used in this study

Primer 16S_F 16S_R agrC_F agrC_R ahpC_F ahpC_R atl_F atl_R cidA_F cidA_R clfA_F clfA_R clfB_F clfB_R clpC_F clpC_R fnbA_F fnbA_R icaA_F icaA_R icaR_F icaR_R rbf_F rbf_R rot_F rot_R saeS_F saeS_R sarA_F sarA_R sarX_F sarX_R sod_F sod_R Sequence GCG CTG CAT TAG CTA GTT GGT TGG CCG ATC ACC CTC TCA CCA GCT ATA ATTAGT GGT ATT AAG TAC AGT AAA CT AGG ACG CGC TAT CAA ACA TTT T GCA TGA CCA TTC AGA TGC AA CCA ATT CCG TCA GCG TTA AT TTT GGT TTC CAG AGC CAG AC TTG GGT TAA AGA AGG CGA TG AAT TTC GGA AGC AAC ATC CA CTT CCC TTA GCC GGC AGT AT TTT CAA CAA CGC AAG ATA GCT ACT GCC GCT AAA CTA TTT GGG ATA GGC AAT CAT CA TCA TTT GTT GAA GCT GGC TC GAA AAA TTA ACG GGC GGA TT GCA ATC TCT CCG AGT GG CCA GGT GGT GGT CAG GTT AC TGT GCT TGA CCA TGC TCT TC CGC ACT CAA AGG CAT T CCA GCA AGT GTC TGA CTT CG CCA AAT TTT TGC GAA AAG GA TAC GCC TGA GGA ATT TTC TG ACG CGT TGC CAA GAT GGC ATA GTC TT AGC CTA ATT CCG CAA ACC AAT CGC TA TCG CTT TCA ATC TCG CTG AA CGA CAC TGT ATT TGG AAT TTT GCA AAT CCA GAA CCA CCC GTT TT ACG CCA CTT GAG CGT ATT TT GCA CAA CAA CGT AAA AAA ATC GAA TTC GTT GTT TGC TTC AGT GAT TC GTC CTA CTT AAA TCT AGC TCA TCC CTG AGA AAT TAG AAA CAT TGC TTG GC CCA ATG TAG TCA GGG CGT TT GTT CAG GTT GGG CTT GGT TA

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Cell culture adherence assay. A549 cells were grown to conuence in 24-well plates. S. aureus was exposed to various concentrations (10, 50, or 100%) of CSE or TSBG alone using the short-term exposure protocol described above, washed in sterile MEM twice, and resuspended in sterile MEM. The nal OD600 was adjusted to 0.45 so as to achieve a multiplicity of infection (MOI) of 10. The inoculum CFU (IC) was enumerated. In order to facilitate the bacterial contact with A549 cells, centrifugation was carried out at 800 g for 5 min at room temperature. After incubation for 1 h at 37C in the presence of 5% CO2, wells were washed with sterile D-PBS with 1 mM Ca2 and 1 mM Mg2 three times to remove nonadherent bacteria. Adherent bacteria (AB) were enumerated and percent adherence frequency calculated as (AB IC) 100. Statistical analysis. The biolm assay results are expressed as averages of quadruplicate readings standard deviations from minimum of 2 different experiments. Data were compared by one-way analysis of variance (ANOVA) followed by Dunnets multiple-comparison test. The gene expression results (see Fig. 2) are expressed as averages of values from triplicate readings standard deviations from one representative out of three experiments. The gene expression data were compared by the twotailed t test. Prism 4 software (GraphPad) was used for all statistical analysis. A P value of less than 0.05 was considered signicant.

RESULTS

CS exposure induces biolm formation in multiple strains of S. aureus. To determine the effects of CS exposure on biolm formation, S. aureus strains were exposed to various concentrations

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FIG 1 CS induces staphylococcal biolm formation. S. aureus strains were exposed to various concentrations of CSE (0, 10, 25, or 50%) for 18 h or to 0 or 100% CSE for a short period (1 h), and biolm formation was quantied. (A) Crystal violet staining of biolm-associated biomass revealed CS-mediated dosedependent upregulation of biolm formation in different strains of S. aureus as indicated. Histograms indicate average values of quadruplicate readings from at least two separate experiments standard deviations. (B) This was further conrmed by enumeration of bacterial CFU in sessile and planktonic fractions at the end of 18 h of incubation, which showed signicantly more sessile than planktonic CFU. Average percent CFU values from three separate experiments standard deviations are shown. (C) CS-induced biolm formation was observed even when S. aureus Newman, RN6390, and USA300 were exposed to CS for a short period (1 h) Averages of quadruplicate readings from at least two separate experiments standard deviations are shown. P values for this and subsequent gures are denoted as follows: *, P 0.05; **, P 0.001; ***, P 0.0001.

(0, 10, 25, or 50%) of CSE for 18 h and biolm formation quantied. Treatment with CSE led to a dose-dependent increase in biolm formation in S. aureus strains Newman, RN6390, and 502A (Fig. 1A). For S. aureus USA300, we observed maximum biolm augmentation following exposure to 10% CSE (Fig. 1A). In order to conrm these results, we plated serial dilutions of planktonic and sessile (biolm-associated) fractions. For S. aureus strains Newman and RN6390, there was a signicantly (P 0.05) higher number of bacterial CFU in the biolm fraction than in the planktonic fraction upon treatment with 50% CSE (Fig. 1B and data not shown.) We noted that even a short-term (1-h) exposure to CS was enough to prime increased biolm formation in S. aureus (Fig. 1C). Short-term exposure to CS did not affect bacterial viability (see Fig. S1 in the supplemental material). CS exposure rapidly alters transcription of genes relevant to biolm formation. The quorum-sensing accessory gene regulator (agr) system controls transition between planktonic and sessile forms of S. aureus (52). Bacteria dispersing from biolms have been demonstrated to exhibit increased agr activity (75). We analyzed whether CS-mediated augmentation of S. aureus biolms was a result of alterations in agr expression by comparing levels of agrC transcripts in S. aureus cells exposed to CS for 1 h. For Newman (Fig. 2A), RN6390 (Fig. 2B), and USA300 (Fig. 2C), 1 h of exposure to CS led to an approximately 10-fold (P 0.01) decrease in the level of agrC compared to that in untreated cells. Staphylococcal accessory regulator A (SarA) is a regulator of

the expression of virulence factors such as FnbA adhesin and -, -, and -toxins (13, 14). It activates biolm formation by increasing transcription of the ica (intercellular adhesin) operon, which is involved in the production of exopolysaccharide adhesin (12). Another gene, rbf (required for biolm formation), induces biolm formation by downregulating the icaR negative regulator (20). Short-term exposure to CSE led to a 10- to 15-fold increase in sarA and rbf transcripts in different S. aureus strains. This effect was not accompanied by induction of icaA or by downregulation of icaR in RN6390 and Newman (see Fig. S2A and B in the supplemental material), while in the case of USA300, we observed an 100-fold increase in icaA transcript levels following short-term exposure to CSE (see Fig. S2C in the supplemental material). CS-mediated enhancement of staphylococcal biolms requires oxidative stress. CS contains many free radicals and reactive oxygen species (such as O2, NO, OH, and H2O2) that increase oxidative stress and may contribute to pathology. qRT-PCR analysis of CS-exposed S. aureus Newman revealed statistically signicant increases in the transcripts of oxidoreductase genes such as ahpC (encoding alkyl hydroxy peroxidase) and sod (encoding superoxide dismutase) (Fig. 2A). To analyze whether CSmediated upregulation of S. aureus biolm is due to the action of reactive oxygen species on bacteria, we pretreated CS with Nacetyl cysteine (NAC). Treatment with 25 mM NAC abrogated the ability of CS to upregulate biolm formation, consistent with a role for the oxidative stress in this phenomenon (Fig. 3A). This

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FIG 2 CS exposure rapidly alters transcription of important oxidoreductase, surface adhesin, and biolm-associated genes. RNA was extracted from S. aureus
Newman (A), RN6390 (B), and USA300 (C) exposed to 100% CSE or TSBG alone for 1 h and reverse transcribed. Transcript levels for relevant genes, as indicated on x axis, were determined using real-time PCR with normalization to 16S rRNA. RQ values were calculated by comparative threshold cycle (CT), following which the fold difference over transcript levels in bacteria exposed to TSBG alone was determined. RQ fold difference values from triplicate readings in one representative experiment (out of at least two separate experiments) standard deviations are shown.

was further corroborated by observation that exposure to H2O2 alone leads to increased production of biolms in Newman and USA300 (Fig. 3B) and in RN6390 (data not shown). We also observed reduced transcription of ahpC and sod and increased levels of agr transcripts in USA300 exposed to NAC-pretreated CS compared to levels from bacteria exposed to CS alone (Fig. 3C). CS-enhanced biolms are DNase and proteinase K sensitive. The staphylococcal biolm matrix has been reported to contain extracellular genomic DNA (eDNA), proteins, and polysaccharides (17, 19, 58). We observed that addition of either DNase I or proteinase K leads to dispersal of established CS-induced biolms, reducing them to the level of biolms formed by untreated staphylococci (Fig. 4). Susceptibility of staphylococcal biolms to DNase I as well as proteinase K is well established. eDNA release has been described to be dependent on cell death and lysis mediated by the cidA murein hydrolase (58). Notably, we observed a minor (1.7-fold) increase in the level of cidA transcripts in CStreated S. aureus Newman (see Fig. S2A in the supplemental material), an 8-fold increase in RN6390 (see Fig. S2B in the supplemental material), and a 6-fold increase in USA300 (see Fig. S2C in the supplemental material) over control values. CS exposure induces S. aureus binding to bronectin and adherence to human cells. Fibronectin binding enhances S. aureus adherence to human cells (47). Moreover, overexpression of surface adhesin FnbA leads to formation of a staphylococcal biolm with a proteinaceous matrix (68). We observed that CS exposure led to increased transcription of fnbA compared to that in control bacteria grown in the absence of CS (Fig. 2). CS-exposed S. aureus RN6390 and USA300 adhered to immobilized human bronectin at signicantly higher levels than bacteria grown in CS-

free medium (Fig. 5A). In contrast, strain Newman exhibited weaker overall bronectin binding (Fig. 5A). Prior work has established that S. aureus Newman is weakly adherent to immobilized bronectin despite robust transcription of fnbA because of a premature stop codon that leads to the production of truncated protein that is not anchored to the bacterial outer membrane (30, 60). Increased binding of CS-exposed Newman to immobilized bronectin may be attributed to a mechanism such as extracellular adhesion protein (Eap) (31), which functions independently of bronectin binding proteins (FnBPs), or to other surface-attached MSCRAMM proteins that may bind human bronectin. CS-treated Newman and USA300 (Fig. 5B) as well as RN6390 (data not shown) adhered to A549 human lung epithelial cells more efciently than control bacteria.
DISCUSSION

The observation that cigarette smokers and individuals exposed to second-hand smoke are more susceptible to upper respiratory tract infections is conventionally attributed to immunomodulatory and other deleterious effects of CS components (61). We hypothesized that CS might have direct effects on members of the human-associated microbiota as well. This theme has been explored by recent publications, including a prospective study showing a strong correlation between CS exposure and increased acquisition of pathogenic bacteria in subgingival plaques (38) and studies showing augmentation of dental plaque-associated biolms owing to increased production of Porphyromonas gingivalis mbrial protein in the presence of CS (2), altered expression of outer membrane protein leading to suppression of the proinammatory cytokine response mediated by P. gingivalis (3), and CS-

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FIG 3 CS induces biolm formation in an oxidant-dependent manner. S. aureus Newman and USA300 were exposed to vehicle control or CS with or without NAC pretreatment for 30 min and used in a biolm assay. (A) Antioxidant treatment abrogated the ability of CS to induce biolm formation, indicating a role for oxidative stress in this phenomenon. (NS, P 0.05.) (B) A similar biolm augmentation phenotype was observed when S. aureus Newman and USA300 were exposed to hydrogen peroxide. Averages of quadruplicate readings from at least two separate experiments standard deviations are shown for panels A and B. (C) USA300 cells were used for qRT-PCR analysis as described for Fig. 2. The gene expression pattern in bacteria exposed to NAC-pretreated CS shows signicantly reduced transcription of oxidoreductase genes (ahpC and sod) and increased transcription of agrC compared to that in bacteria exposed to CS alone. (***, P 0.0001 compared to medium alone; ###, P 0.001 compared with CS-treated bacteria).

mediated induction of biolm formation in Streptococcus pneumoniae (51) and Pseudomonas aeruginosa (1). Results from different epidemiological studies to determine the nature of the association between smoking status and staphylococcal preva-

FIG 4 Extracellular DNA and proteins are important constituents of the CSinduced staphylococcal biolm matrix. S. aureus Newman and USA300 cells were exposed to 100% CSE or medium alone for 1 h, CSE was washed off, and bacteria were grown in 0.5% TSBG for 18 h in 96-well polystyrene plates. Biolms were treated with DNase I or proteinase K for 3 h and biolm formation quantied. DNase I as well as proteinase K treatment caused dispersal of CS-induced biolms. Average A590 values of triplicate readings from three separate experiments standard deviations are shown.

lence are inconclusive: Olsen et al. attributed decreased S. aureus nasal carriage in smokers to the bactericidal activity of CS components and increased immune activity due to smoke-induced hypoxia (53), Wang et al. found no signicant association between smoking and acquisition of community-acquired MRSA (70), and Bogaert et al. and Melles et al. observed that in children (18 years of age), exposure to passive (and not active) smoking increases S. aureus nasal carriage (8, 46). We observed that CS exposure led to increased biolm formation and enhancement of bronectin binding in an important human pathogen, S. aureus. Biolms are surface-attached multicellular communities enclosed inside a polymeric matrix, the bacterial residents of which exhibit coordinated behavior (21, 52). S. aureus can adhere to a variety of host tissues and establish biolms that are encased within a matrix composed of extracellular polysaccharides (17), proteins (19), and eDNA (58). In this work, we observed upregulation of biolm formation when different S. aureus strains, including important clinical isolates such as USA300, were exposed to CS. These ndings are consistent with the observations made by Goldstein-Daruech et al. (27), who demonstrated that in bacteria isolated from sinonasal cavities of smokers suffering from chronic rhinosinusitis (CRS), biolm formation is increased upon in vitro exposure to CS, while in bacteria derived from a nonsmoking CRS population, CS-mediated upregulation of biolm formation is absent. We noted that even a short (1-h) exposure followed by removal of CS was enough to commit S. aureus to a sessile, biolmassociated life style.

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FIG 5 CS enhances binding to bronectin and adherence to human cells. S. aureus treated with CS or vehicle control was assayed for binding to bronectincoated wells (A) or A549 cells (B). CS-exposed Newman, RN6390, and USA300 adhere at signicantly (P 0.0001) higher levels than their untreated counterparts. For bronectin binding, the averages of sextuplicate readings from at least two separate experiments standard deviations are shown. For A549 cell adherence, the average percent adherence for triplicate wells from two separate experiments standard deviation is shown.

Two distinct molecular pathways leading to biolm formation have been described in S. aureus. The ica-dependent pathway involves enzymes for the synthesis of exopolysaccharide (poly-Nacetylglucosamine) adhesin (17). In contrast, ica-independent pathways employ surface-anchored protein adhesins such as biolm-associated protein (Bap) of mastitis-associated staphylococci (19), -toxin (69), and other factors that increase membrane hydrophobicity, such as teichoic acids (28), to form proteinaceous biolms. The ica operon is regulated negatively by icaR (34) and positively by sarA (encoding staphylococcal accessory regulator) (5), sarX (20), and rbf (43). We noted marked upregulation of sarA and rbf transcription in CS-treated bacteria (Fig. 2). The quorum-sensing agr system is another important coordinator of gene expression that plays a crucial role in S. aureus biolm dispersal by controlling the production of surfactants such as phenol-soluble modulins (55, 75). Real-time PCR analysis revealed an 10-fold reduction in the levels of agrC transcript in S. aureus cells treated with CS for 1 h compared to that in cells grown in the absence of CS. The agr quorum-sensing system is involved in the establishment of staphylococcal infections, as it controls production of virulence factors such as hemolysins that aid the pathogen to evade the host immune response. Biolm formation is favored by conditions that reduce agr activity (9), and agr expression remains low except in the areas of bacterial cells that are detaching from the biolms (75). It must be noted that while the expression patterns for most of the genes were similar for the different staphylococcal strains, there were some obvious differences that may be attributed to intrinsic genetic differences in these strains (7). For exam-

ple, clpC, cidA, and clfA were expressed approximately 2-, 8-, and 9-fold, respectively, in CS-treated RN6390, 3-, 2-, and 1.2-fold, respectively, in Newman, and 6-, 0.3-, and 0.06-fold, respectively, in USA300. The high concentrations of a variety of free radicals and reactive oxygen species, such as nitrous oxide (NO), superoxide anions, hydroxyl radicals, and hydrogen peroxide, present in the CS alters important signaling pathways, leading to immunomodulatory effects (6, 35, 44, 45, 64, 67). The qRT-PCR analysis of staphylococci treated with CS showed signicant increases in the transcript levels of important oxidoreductases such as ahpC and sod. S. aureus employs SodA and SodM to catalyze the conversion of O2 to O2 and H2O2 (36); following these reactions, the enzymes catalase and Ahp reduce H2O2 to H2O and O2 (16). Pretreatment of CS with an antioxidant compound (NAC) signicantly abrogated induction of staphylococcal biolm formation. Using qRT-PCR, we also conrmed that in staphylococci exposed to NAC-pretreated CSE, transcript levels of oxidoreductase genes ahpC and sod were lower than what was seen in bacteria treated with CSE alone. This was accompanied by increased levels of agrC transcripts in staphylococci exposed to NAC-pretreated CSE. Moreover, sublethal levels of hydrogen peroxide alone enhanced biolm formation. The previously reported inhibition of S. aureus biolms by hydrogen peroxide at concentrations of 2.5% may be attributed to its bactericidal activity, as the 50% inhibitory concentration (IC50) of H2O2 for S. aureus is 500 mM (1.7%) (66, 76). We propose that at the substantially lower concentrations used in our studies (5 mM [0.015%] to 10 mM [0.03%]), H2O2

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may induce oxidative stress conditions akin to those generated by exposure to CSE without being toxic to S. aureus cells. These observations imply a cross talk between the oxidative stress response and the quorum-sensing agr system resulting in CS-mediated upregulation of biolm formation in S. aureus. Indeed, these observations are in accordance with a recently published report by Sun et al. (63) showing that the agr system of staphylococci has a built-in oxidation-sensing mechanism in the form of an intramolecular disulde switch in AgrA. Oxidative stress induces disulde bond formation in AgrA, rendering it unable to bind the agr regulon promoter (P2) and in turn reducing the transcription of agrC (63). Other gene products that are involved in the formation of biolms include murein hydrolase CidA (58) and Clp proteases (26). This process is further complicated by the fact that biolm formation and the composition of the biolm matrix may also be dependent on a variety of environmental signals, which include osmolarity (39), available glucose (39), anaerobiosis (28), and levels of iron, copper (4), nitrites, citrate, and ethanol. Staphylococcal biolms induced by CS exposure were observed to be susceptible to enzymatic treatment with DNase I and proteinase K, similar to what is seen in bacteria grown in the absence of CS, indicating that eDNA and proteins may be important structural components of CS-induced staphylococcal biolms. CS exposure also increased fnbA transcription and staphylococcal binding to bronectincoated wells in a dose-dependent manner. Fibronectin is an important extracellular matrix protein found in plasma and on cell surfaces. Binding to bronectin increases S. aureus adherence and invasion and may be relevant to both colonization and invasive disease (23). Environmental stimuli such as CS may have profound impacts on human cells, with implications for a wide variety of disease processes. As our appreciation for the pleiotropic effects of the human-associated microbiota on health grows (40), it is important to remember that our resident microbes may share our exposures through gastrointestinal, respiratory, or other modes of entry. Such exposures may alter microbial physiology in ways relevant to human health. Because of the prevalence of cigarette smoking and the relevance of S. aureus to health, we explored one such potential interaction. CS exposure markedly alters S. aureus gene expression as well as phenotypic characteristics relevant to pathogenesis. Conrmation of these ndings in relevant animal models of colonization and disease is warranted.
ACKNOWLEDGMENTS
We thank Barry N. Kreiswirth (Public Health Research Institute, UMDNJ) for the kind gift of S. aureus strain 502A. This work was supported by the Flight Attendant Medical Research Institute (grants YCSA_092417 to R.K. and CIA_092081 to A.J.R.).

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