**3. E‐cigarette vapor increases staphylococcal virulence and impairs innate immune function**

As the popularity of e‐cigarettes skyrockets, it becomes increasingly important to establish whether e‐cigarettes are indeed a safer alternative to conventional cigarettes. Simply, it is a battery operated device designed to deliver nicotine with flavors and other chemicals to users in vapor form instead of smoke. E‐cigarette vapor (EV) contains a far more limited array of chemicals than cigarette smoke, resulting from vaporization of e‐liquid containing nicotine, propylene glycol (PG), and vegetable glycerin (VG) solvents, with different flavors added in some cases. While PG is not harmful when ingested through the GI tract and is generally recognized as safe for use as a food additive [27], high wattage heating of these solvents results in the production of formaldehyde, aldehyde, and acrolein, resulting in significantly increased exposure of the airways to formaldehyde relative to a pack‐day smoker [28]. The absorption of nicotine in blood showed no difference between the latest generation e‐cigarettes and conventional cigarettes. Nicotine itself is toxic at high doses, and as described in the preceding section, induces a shift in MRSA toward a less negative surface charge which confers resistance to AMPs. Thus, e‐cigarette "vaping" is exposing the colonizing bacteria to at least two components (formaldehyde and nicotine) with the potential to induce a significant stress response.

#### **3.1. MRSA growth suppression by e‐cigarette vapor**

We tested the growth kinetics of MRSA cultures during exposure to different components of EV extract (EVE) including nicotine, PG, and VG. We also tested a number of different brands of e‐cigarettes. EVE suppressed MRSA growth, with cultures failing to achieve logarithmic phase. While nicotine mildly inhibited MRSA growth in a dose‐dependent fashion, the vaporized vehicles likely account for most of the suppressive effects observed. Vaporized PG and/ or VG nearly abrogated MRSA growth, to the same degree as EVE [29]. Interestingly, MRSA growth suppression was observed in four of five brands, suggesting that the etiologic agent is a common component across brands. Thus, as with cigarette smoke, EV imposes significant stress on Staphylococcal cells. Bacterial survivors of the noxious exposure are likely to be hardier, and thus more difficult to kill.

#### **3.2. Increased hydrophobicity, adherence, and invasion of keratinocytes by e‐cigarette vapor exposed MRSA**

As with conventional CS, EV promotes a shift in phenotype that supports persistent MRSA colonization of the epithelium, as well as increasing the risk of invasive infection. Hydrophobicity was markedly increased following EV exposure (by 31%), with a corresponding doubling of MRSA adherence to the human keratinocyte HaCaT cell line. EVE‐exposed MRSA demonstrated increased invasion of and intracellular persistence within HaCaT cells. These changes do not appear to be induced by nicotine, as increasing the nicotine content by fivefold had no effect on adherence and invasion.

#### **3.3. E‐cigarette vapor increases MRSA resistance to human antimicrobial peptide LL‐37**

Increased survival of internalized MRSA suggests that e‐cigarette exposure may induce resistance to killing by epithelial cells. This effect is at least in part a consequence of moderately increased resistance to killing by antimicrobial peptides (AMPs). One of the established mechanisms for bacterial virulence is alterations in surface charge. Most bacteria have predominantly anionic surfaces, which are targeted by the human innate immune system [30, 31]. One of the human AMPs, LL‐37, acts through charge interactions between its cationic surface and the bacterial lipids which are negatively charged [30–32]. EVE exposure increased the MIC of LL‐37 from 7 uM in unexposed MRSA to 10 uM in EVE‐MRSA (P = 0.014). As with conventional CS, EVE exposure induces a shift in MRSA toward a less negative surface charge. In turn, this reduces the propensity of the cationic LL‐37 to bind the bacterial surface as it must for antimicrobial activity. This transition in surface charge was independent of the level of nicotine in EVE.

#### **3.4. MRSA biofilm induction by e‐cigarette vapor**

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

**3. E‐cigarette vapor increases staphylococcal virulence and impairs innate** 

As the popularity of e‐cigarettes skyrockets, it becomes increasingly important to establish whether e‐cigarettes are indeed a safer alternative to conventional cigarettes. Simply, it is a battery operated device designed to deliver nicotine with flavors and other chemicals to users in vapor form instead of smoke. E‐cigarette vapor (EV) contains a far more limited array of chemicals than cigarette smoke, resulting from vaporization of e‐liquid containing nicotine, propylene glycol (PG), and vegetable glycerin (VG) solvents, with different flavors added in some cases. While PG is not harmful when ingested through the GI tract and is generally recognized as safe for use as a food additive [27], high wattage heating of these solvents results in the production of formaldehyde, aldehyde, and acrolein, resulting in significantly increased exposure of the airways to formaldehyde relative to a pack‐day smoker [28]. The absorption of nicotine in blood showed no difference between the latest generation e‐cigarettes and conventional cigarettes. Nicotine itself is toxic at high doses, and as described in the preceding section, induces a shift in MRSA toward a less negative surface charge which confers resistance to AMPs. Thus, e‐cigarette "vaping" is exposing the colonizing bacteria to at least two components (formaldehyde and nicotine) with the potential to induce a signifi-

We tested the growth kinetics of MRSA cultures during exposure to different components of EV extract (EVE) including nicotine, PG, and VG. We also tested a number of different brands of e‐cigarettes. EVE suppressed MRSA growth, with cultures failing to achieve logarithmic phase. While nicotine mildly inhibited MRSA growth in a dose‐dependent fashion, the vaporized vehicles likely account for most of the suppressive effects observed. Vaporized PG and/ or VG nearly abrogated MRSA growth, to the same degree as EVE [29]. Interestingly, MRSA growth suppression was observed in four of five brands, suggesting that the etiologic agent is a common component across brands. Thus, as with cigarette smoke, EV imposes significant stress on Staphylococcal cells. Bacterial survivors of the noxious exposure are likely to be

**3.2. Increased hydrophobicity, adherence, and invasion of keratinocytes by e‐cigarette** 

As with conventional CS, EV promotes a shift in phenotype that supports persistent MRSA colonization of the epithelium, as well as increasing the risk of invasive infection. Hydrophobicity was markedly increased following EV exposure (by 31%), with a corresponding doubling of

diseases.

**immune function**

110 Frontiers in Frontiers in Staphylococcus Aureus *Staphylococcus aureus*

cant stress response.

**3.1. MRSA growth suppression by e‐cigarette vapor**

hardier, and thus more difficult to kill.

**vapor exposed MRSA**

EV has other pro‐virulent effects on MRSA that may allow the bacteria to cause more severe infections—it promotes a moderate increase in biofilm formation. Antibiotics and our own immune cells have difficulty penetrating biofilm to kill embedded bacteria, thus allowing colonization (or infection) to perpetuate. The increased predilection for biofilm following EV exposure may reflect a response to nicotine in the vapor, as nicotine alone induced dose‐ dependent increases in biofilm formation. Interestingly, the increased biofilm formation occurring in response to conventional CS seemed to be due primarily to oxidative stress [29]. In our studies, MRSA incubated with only nicotine produced more biofilm than control. In the exposure to either EV or CS, a novel pathway in inducing MRSA biofilm was suggested [23, 29].

#### **3.5. E‐cigarette vapor increases MRSA virulence in a mouse model of pneumonia**

*In vivo* studies in mice also suggested that EV promotes propathogenic features. The same murine pneumonia model used to evaluate CS effects on MRSA was employed to determine EV effects. Mortality was increased in mice infected with EV‐MRSA (25 vs. 0% in mice infected with control MRSA, *p* < 0.05). In addition, bacterial burdens were 10‐fold higher in mice infected with EV‐MRSA relative to mice infected with MRSA controls. Exposure to EV enhanced both MRSA virulence and MRSA survival [29].

#### **3.6. E‐cigarette vapor induction of MRSA virulence gene expression**

Similarly to the effect of CS exposure on MRSA virulence, MRSA exposed to EV was more virulent than controls in the biological model. Thus, the expression of several well‐known virulence factors after EV exposure was evaluated. Panton‐Valentine leukocidin (*pvl*), α‐ hemolysin (*hla*), coagulase (*coa*), α‐phenol soluble modulin (*psm‐α*), intracellular adhesion (*icaA*), staphylococcal protein A (*spa*), and quorum sensing (*agrA*) were quantified relative to 16s rRNA as a housekeeping gene. After EV exposure, the expression of *coa* and *pvl* increased by 1.68‐ and 1.56‐fold, respectively [29]. Expression of *spa* did not change with EV exposure while *icaA, agrA, hla,* and *psm* decreased.
