Cessation from Smoking Improves Innate Host Defense and Clearance of Experimentally Inoculated Nasal Staphylococcus aureus

Abstract

Staphylococcus aureus nasal carriage is transient in most humans and usually benign, but dissemination of S. aureus to extranasal sites causes the majority of clinical infections, and S. aureus is a major cause of serious infections in the United States. A better understanding of innate nasal decolonization mechanisms is urgently needed, as are relevant models for studying S. aureus clearance. Here, we screened a population of healthy smokers for nasal S. aureus carriage and compared the participants' abilities to clear experimentally applied nasal S. aureus before and after completion of a smoking cessation program. We determined that cigarette smoking increases the mean nasal S. aureus load (2.6 × 10⁴ CFU/swab) compared to the load observed in healthy nonsmokers (1.7 × 10³ CFU/swab) and might increase the rate of S. aureus nasal carriage in otherwise-healthy adults: 22 of 99 smokers carried S. aureus at the screening visit, while only 4 of 30 nonsmokers screened positive during the same time period. Only 6 of 19 experimental inoculation studies in active smokers resulted in S. aureus clearance within the month of follow-up, while in the cessation group, 6 of 9 subjects cleared nasal S. aureus and carriage duration averaged 21 ± 4 days. Smoking cessation associated with enhanced expression of S. aureus-associated interleukin-1β (IL-1β) and granulocyte colony-stimulating factor (G-CSF) in nasal fluids. Participants who failed to clear S. aureus exhibited a higher nasal S. aureus load and elevated nasal interleukin-1 receptor antagonist (IL-1RA) expression at the preexperiment study visits. We conclude that smokers exhibit higher S. aureus loads than nonsmokers and that innate immune pathways, including G-CSF expression and signaling through the IL-1 axis, are important mediators of nasal S. aureus clearance.

Keywords: Staphylococcus aureus; experimental colonization; host defense; humans; nasal carriage; smoking cessation.

FIG 1

Cigarette smoking associates with elevated natural nasal S. aureus load. (A) Nasal S. aureus load (average S. aureus CFU/swab for two nostrils) at the screening visit of healthy smokers (solid black circles) versus the average nasal S. aureus load per S. aureus-positive visit for nonsmokers (open circles) who were monitored longitudinally during the same time period. Error bars represent the means and standard errors of the means (SEM). (B) Seasonal nasal S. aureus load for smoker screening visits (bold X's) versus the average nasal S. aureus load for 14 nonsmoker carriers (open circles). Solid lines enveloping the open circles indicate the SEM for nonsmoker average nasal S. aureus load. The dotted horizontal line in each panel indicates the S. aureus detection limit for nasal swab samples (20 CFU/swab).

FIG 2

Experimental design and timeline. Healthy smokers were screened for nasal S. aureus (SA) carriage, and S. aureus isolates were genotyped and prepared as frozen stocks for subsequent inoculations. Participants applied topical nasal mupirocin twice daily for 5 days (days −17 to −13, preinoculation) to clear endogenous nasal S. aureus. On day −6 (preinoculation), nasal swabs were collected to confirm S. aureus decolonization, and nasal secretions were collected. On days 0 and 1, participants were swabbed for assessment of S. aureus carriage status and self-applied 2 × 10⁷ CFU of their own S. aureus strain to each nostril. Nasal swabs were collected every 3 to 4 days postinoculation. Nasal secretions were collected at days 3, 8, 11, 15 or 16, 23 or 24, and 31 or 32 postinoculation. Following the month of follow-up after S. aureus inoculation, participants attended a 6-week Quit Smoking Now (QSN) cessation course. Subjects who successfully quit smoking and maintained cessation for an additional 4 weeks repeated the decolonization/recolonization protocol.

FIG 3

Cessation from smoking associates with a higher rate of S. aureus clearance during experimental inoculation studies. (A) Comparison of experimental S. aureus carriage duration between all subjects who were actively smoking and those who recently quit smoking. (B) Average expired-air carbon monoxide (CO) values for participants who reported smoking compared to those who reported cessation of cigarette smoking. (C) Pairwise comparison of experimental nasal S. aureus carriage duration for 8 participants (each represented by a unique symbol) who completed both study arms. Open symbols indicate participants who failed to clear S. aureus in one or both study arms. Solid symbols indicate participants who cleared experimentally inoculated S. aureus in one or both study arms. ns, not significant.

FIG 4

Decreased natural nasal S. aureus load in subjects who successfully quit smoking. Nasal S. aureus loads (average S. aureus CFU/swab for both nostrils) at day −17 (preexperiment) of the smoking and cessation study arms. Error bars represent the means and SEM. The dotted horizontal line indicates the limit of detection for nasal S. aureus (20 CFU/swab).

FIG 5

Smoking cessation associates with augmented innate nasal mucosal defense against S. aureus. Participants were cleared of endogenous nasal S. aureus and subsequently reinoculated with their own S. aureus strain as described for Fig. 2. Nasal fluids were collected from each participant at day −17 (prior to mupirocin application for clearance of endogenous S. aureus), day −6 (one week after the last mupirocin application), and at days 3, 8, 11, 15 or 16, 22 or 23, and 31 or 32 postinoculation with autologous S. aureus. Fluids were analyzed using Bio-Rad's 27-plex proinflammatory panel, and results are shown for G-CSF (A), IL-1β (B), and the Il-1RA-to-Il-1β ratio (C), where gray circles represent nasal fluids collected from S. aureus-negative noses (n = 34 for smoking, n = 27 for cessation groups) and black circles represent nasal fluids collected from S. aureus-positive noses (n = 100 for smoking, n = 33 for cessation groups). Error bars represent the means and SEM.

FIG 6

Clearance of experimentally inoculated nasal S. aureus correlated with nasal S. aureus load at screening and elevated IL-1RA in nasal secretions. (A) Nasal S. aureus levels at the screening and day −17 (premupirocin) visits of participants who eventually cleared (“Clearers”) or carried (“Carriers”) experimentally inoculated S. aureus. The dotted horizontal line indicates the limit of detection. (B) Participants who carried experimentally inoculated S. aureus (“Carriers”) exhibited elevated natural (day −17/premupirocin) expression of IL-1RA compared to participants who cleared (“Clearers”). In panels A and B, error bars represent the means and SEM. (C) Positive correlation between natural nasal S. aureus load (x axis) and IL-1RA expression in nasal fluid (y axis) collected from subjects at day −17.