**2. Conventional cigarette smoke promotes increased staphylococcal virulence**

MRSA often colonizes the nasopharynx, where it is exposed to everything that an individual inhales, including cigarette smoke (CS) or e‐cigarette vapor (EV). Bacteria from the nasopharynx have the potential to travel to the lungs, causing pneumonia, or to the skin, where compromises in the epithelial barrier such as ulcers, abrasions, or surgical incisions provide ports of entry for invasive staphylococcal infection. The initial stage preceding infection is colonization of the human host. Transient colonization of the nasopharynx by *S. aureus* is more common in healthy individuals than persistent colonization (30 vs. 20%), which usually entails a larger bacterial burden and increased risk of infection [15]. Smokers are known to have higher rates of persistent colonization of the nasopharynx by MRSA relative to the general population [17]. The phenotypic changes in *S. aureus* induced by *in vitro* CS exposure favor persistent colonization via increasing adhesion to host cells and by inducing biofilm formation, reducing the ability of host immune effectors to limit colonization [22].

#### **2.1. MRSA hydrophobicity changes induced by cigarette smoke**

Our own studies demonstrate that CS extract (CSE) exposure results in a dose‐dependent increase in MRSA surface hydrophobicity that is persistent and possibly even heritable [23, 24]. Increased hydrophobicity is associated with increased bacterial interactions with host epithelial cells [25]. Thus, we evaluated the adherence of MRSA to human keratinocytes (HaCaT cells) *in vitro* and found that pretreatment with CSE increased adherence from 28% (control) to 52% (CSE‐MRSA). These findings are likely due to increased interaction with  epithelial cell surfaces because of the increased surface hydrophobicity induced by CSE exposure. Increased capacity to adhere to epithelial cells as a consequence of CS exposure may explain the increased MRSA carriage rates in smokers.

#### **2.2. Staphylococcal surface charge changes induced by cigarette smoke**

CSE exposure induced dramatic changes in surface charge, in a dose‐dependent manner. With increasing concentrations of CSE, the surfaces of *S. aureus* and MRSA became less negative. The negative surface charge of bacteria is protective against a variety of host defense mechanisms, including antimicrobial peptide (AMP) binding. Changes in surface charge were persistent for over 24 h post‐CSE exposure. These changes suggest that the CSE exposure may result in persistent, heritable changes. Additionally, daily intermittent exposure to CSE over the course of 3 or 4 days—reflecting the pattern of exposure experienced by the nasopharyngeal flora of a smoker—resulted in an additive increase in surface charge alterations.

Cigarette smoke contains thousands of compounds, making the identification of specific etiologic agent inducing change in surface charge difficult. However, we hypothesized that reactive species such as free radicals and carbon monoxide likely played a role. To assess the contribution of reactive species, CSE was stored for 24 h at 4°C to allow time for volatile gases to evaporate and for some degradation of reactive oxygen species. The "aged" CSE induced a less‐anionic surface charge in exposed MRSA; however, it had significantly less potency relative to fresh CSE. Thus, while reactive species account for some measure of the observed surface changes in MRSA, some of the more stable elements of CS contribute as well. One such component, present in both fresh and aged CSE—as well as e‐cigarette vapor—is nicotine. We found that nicotine at 3 and 6 mg/mL induced a dose‐dependent shift toward a less negative surface charge, suggesting that it likely plays a role in the altered surface charge of CSE‐exposed MRSA. Finally, we sedimented CSE and exposed MRSA solely to these particulates and found no effect on surface charge.

The cell surface charge changes are mediated in part by expression of the *mprF* gene, which encodes a membrane protein involved in the shift to a less negative surface charge. Exposure to CSE induced a 1.8‐fold increase in expression of *mprF* RNA, and CSE did not induce significant surface charge change in an *mprF* knock‐out strain of *S. aureus* (SA113 ∆*mprF*), relative to wild‐type bacteria, further supporting an important role of this pathway in this aspect of the staphylococcal response to CS exposure.

#### **2.3. Cigarette smoke exposure increases** *S. aureus* **resistance to antimicrobial defenses**

Aspiration of nasopharyngeal colonizers is a common occurrence and provides an opportunity for potential pathogens to cause airway infection. Numerous immunologic defenses are in place to protect against this outcome, including mechanical clearance of bacteria by the mucociliary elevator and destruction of remaining bacteria by immune cells within the airways. Alveolar macrophages, which destroy bacteria by mechanisms including phagocytosis, antimicrobial peptide (AMP) production, and respiratory burst, are an important component of host defense against pulmonary infection by bacteria.

CSE exposure may induce MRSA resistance to the bacteriocidal activities of these cells by multiple mechanisms. *In vitro*, CSE‐exposed MRSA was resistant to killing by a murine alveolar macrophage cell line (MH‐S), with a fourfold increase in survival relative to control (*p* < 0.0001). This outcome was not due to a cytotoxic effect on the macrophages, as macrophage cell death rate was comparable regardless of whether they were infected with CSE‐treated MRSA or control bacteria. Phagocytosis of fluorescently tagged MRSA‐GFP was unaffected by CSE exposure—suggesting that the difference in survival reflected that the CSE‐exposed MRSA were better able to endure intracellular killing mechanisms including exposure to antimicrobial peptides and other toxic compounds and reactive oxygen species found in the phagolysosome. Indeed, CSE exposure increased resistance to killing by the human AMP LL‐37, with twofold increases in both MIC and MBC relative to control bacteria. Decreased susceptibility to AMPs, which are produced by neutrophils, macrophages, and epithelial cells, suggests a significant increase in pathogenicity.

 epithelial cell surfaces because of the increased surface hydrophobicity induced by CSE exposure. Increased capacity to adhere to epithelial cells as a consequence of CS exposure may

CSE exposure induced dramatic changes in surface charge, in a dose‐dependent manner. With increasing concentrations of CSE, the surfaces of *S. aureus* and MRSA became less negative. The negative surface charge of bacteria is protective against a variety of host defense mechanisms, including antimicrobial peptide (AMP) binding. Changes in surface charge were persistent for over 24 h post‐CSE exposure. These changes suggest that the CSE exposure may result in persistent, heritable changes. Additionally, daily intermittent exposure to CSE over the course of 3 or 4 days—reflecting the pattern of exposure experienced by the nasopharyngeal flora of a smoker—resulted in an additive increase in surface charge

Cigarette smoke contains thousands of compounds, making the identification of specific etiologic agent inducing change in surface charge difficult. However, we hypothesized that reactive species such as free radicals and carbon monoxide likely played a role. To assess the contribution of reactive species, CSE was stored for 24 h at 4°C to allow time for volatile gases to evaporate and for some degradation of reactive oxygen species. The "aged" CSE induced a less‐anionic surface charge in exposed MRSA; however, it had significantly less potency relative to fresh CSE. Thus, while reactive species account for some measure of the observed surface changes in MRSA, some of the more stable elements of CS contribute as well. One such component, present in both fresh and aged CSE—as well as e‐cigarette vapor—is nicotine. We found that nicotine at 3 and 6 mg/mL induced a dose‐dependent shift toward a less negative surface charge, suggesting that it likely plays a role in the altered surface charge of CSE‐exposed MRSA. Finally, we sedimented CSE and exposed MRSA solely to these particu-

The cell surface charge changes are mediated in part by expression of the *mprF* gene, which encodes a membrane protein involved in the shift to a less negative surface charge. Exposure to CSE induced a 1.8‐fold increase in expression of *mprF* RNA, and CSE did not induce significant surface charge change in an *mprF* knock‐out strain of *S. aureus* (SA113 ∆*mprF*), relative to wild‐type bacteria, further supporting an important role of this pathway in this aspect of the

**2.3. Cigarette smoke exposure increases** *S. aureus* **resistance to antimicrobial defenses**

Aspiration of nasopharyngeal colonizers is a common occurrence and provides an opportunity for potential pathogens to cause airway infection. Numerous immunologic defenses are in place to protect against this outcome, including mechanical clearance of bacteria by the mucociliary elevator and destruction of remaining bacteria by immune cells within the airways. Alveolar macrophages, which destroy bacteria by mechanisms including phagocytosis, antimicrobial peptide (AMP) production, and respiratory burst, are an important component

explain the increased MRSA carriage rates in smokers.

lates and found no effect on surface charge.

staphylococcal response to CS exposure.

of host defense against pulmonary infection by bacteria.

alterations.

108 Frontiers in Frontiers in Staphylococcus Aureus *Staphylococcus aureus*

**2.2. Staphylococcal surface charge changes induced by cigarette smoke**

Potential mechanisms of this resistance include changes in cell surface charge and decreased rate of cell division induced by CS. Rapidly dividing bacteria are more sensitive to many antimicrobials, including conventional antibiotics and AMPs. Kristian et al. showed that growth suppression using bacteriostatic antibiotics allowed *S. aureus* decreased susceptibility to killing by AMPs [26]. Growth curves at various concentrations of CSE‐demonstrated inhibition of bacterial growth in a CSE dose‐dependent fashion. The growth defect induced by CSE resolved with removal of CSE; however, it is possible that a clonal population continues to divide slowly and that this population is better equipped to persist in the face of subsequent stressors.

Interestingly, the changes induced by CSE exposure also seemed to induce resistance to membrane solublization by detergent. CSE‐MRSA had similar death rates to control bacteria during the first few minutes of incubation with detergent (Triton‐X), but demonstrated improved survival subsequently, suggesting further protective cell membrane changes induced by CSE.

Findings *in vivo* in a murine model of pneumonia supported *in vitro* results, with CSE exposure resulting in increased staphylococcal persistence in the lungs and an increase in overwhelming infection. Mice infected with CSE‐exposed MRSA had increased bacterial burdens in the lungs at 8 and 24 h relative to those infected with control bacteria, demonstrating that CSE‐MRSA are better able to persist in the face of pulmonary immune defenses. Additionally, 40% of mice infected with a higher inoculum of CSE‐exposed MRSA died within 48 h, vs. only 10% of those infected with unexposed MRSA.

#### **2.4. Potential consequences of virulence changes induced by cigarette smoke**

While pulmonary infection with MRSA is devastating, much of the morbidity and mortality associated with MRSA infections is due to soft tissue infections. Because bacteria colonizing the nasopharynx do not necessarily remain there, propathogenic changes induced by CS in the nasopharynx are of concern for staphylococcal infections beyond the pulmonary system. Coughing, sneezing, etc. lead to significant opportunity to transfer MRSA from nasal passages and upper airway to the skin, where an increased capacity to adhere to and invade epithelial cells—as well as resistance to AMPs—reflect serious potential consequences of the pro‐pathogenic effects of cigarette smoke for skin and soft tissue infections. Finally, MRSA are transmitted between humans in the community as well. Thus, more virulent strains generated by CS exposure may put the community at higher risk of invasive staphylococcal diseases.
