Objective
To determine the effect of smoking cessation on the frequency of recovery of potential pathogens and aerobic and anaerobic interfering bacteria in the nasopharynges of smokers.
Design
Nasopharyngeal cultures were taken from 20 smokers before and 12 to 15 months after cessation of smoking. Potential pathogens and aerobic and anaerobic bacteria with interfering capabilities against these organisms were identified.
Results
Eleven potential pathogens (0.92 pathogens per subject) were isolated from nasopharyngeal cultures obtained from 9 individuals before smoking cessation, and 2 (0.17 per subject) were recovered from 2 individuals after smoking cessation (P<.05). Bacterial interference between 2 aerobic (α and nonhemolytic streptococci) and 2 anaerobic species (Prevotella and Peptostreptococcus species) and 4 potential pathogens (Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pyogenes) was observed. Bacterial interference was noted in 35 instances against the 4 potential pathogens by 14 normal flora isolates that were recovered from the smokers before cessation and in 116 instances by 42 isolates after cessation (P<.01).
Conclusion
To our knowledge, these findings illustrate for the first time that the high number of pathogens and low number of interfering organisms found in the nasopharynx of smokers revert to normal levels after complete cessation of smoking.
Smoking is associated with an increased risk of respiratory tract infection in adults1 and also with oral colonization by some potentially pathogenic microorganisms.2-4 In children, exposure to cigarette smoke is a risk factor for respiratory tract infection and meningococcal meningitis.5 Active smoking and passive exposure to cigarette smoke is also associated with carriage of potentially pathogenic species of bacteria in both adults and children6 possibly owing to enhanced bacterial binding to epithelial cells of smokers7 and the low number of α-hemolytic streptococci with inhibitory activity against Streptococcus pyogenes in the oral cavity of smokers.8 The nasopharyngeal flora of smokers contain fewer aerobic and anaerobic organisms with interfering capability and more potential pathogens compared with those of nonsmokers.9
The purpose of this study was to determine the effect of smoking cessation on the frequency of recovery of potential pathogens and aerobic and anaerobic interfering bacteria in the nasopharynx of smokers.
Twenty healthy adults who had smoked at least 10 cigarettes a day for the past 5 years and had completely ceased smoking were included in the study. None were immune deficient or had any chronic illness such as otitis, sinusitis, or tonsillitis; had received antimicrobial therapy; or had had a respiratory tract infection in the past 3 months prior to the time when the first and second culture samples were taken. The mean age of the patients was 34 years (range, 21-52 years), and 8 were men.
Two culture samples were obtained from each individual: 1 sample before cessation of smoking and the other 12 to 15 months after they stopped smoking. The culture specimens were taken using sterile calcium alginate swabs and were collected from the retropharynx (through the mouth), and were immediately plated into media supportive of the growth of aerobic and anaerobic bacteria. The protocol was approved by the institutional review board.
Sheep's blood (5%), chocolate, and MacConkey agar plates were inoculated for the isolation of aerobic organisms. The culture plates were incubated aerobically at 37°C (MacConkey agar) and under 5% carbon dioxide (blood and chocolate agars), and they were examined at 24 and 48 hours. For the recovery of anaerobic bacteria, the specimens were inoculated onto prereduced vitamin K1–enriched Brucella blood agar, blood agar that contained kanamycin sulfate and vancomycin, and an aerobic blood plate that contained phenylethyl alcohol and enriched thioglycolate broth. These media were immediately incubated in anaerobic containers (Gas Pack; Baltimore Biological Laboratories, Cockeysville, Md) at 37°C and examined after 48 and 96 hours of incubation at 37°C. All types of colonies on each plate were isolated. The mean total number of aerobic and anaerobic bacterial isolates processed from each individual was 7.8 (range, 4-17). Aerobic and anaerobic bacteria were identified by previously described methods.10
The inhibitory activity was tested in a blind fashion, against 1 strain each of a recent clinical isolate of Streptococcus pneumoniae, Haemophilus influenzae (non–type b), Moraxella catarrhalis, and S pyogenes. Inhibitory activity of 5 separate colonies of all aerobic and anaerobic isolates was evaluated. The inhibitory activity of each isolate was individually tested against the test organisms, using the Steer steel pin replicator as previously described.11 In brief, minidrops of log-phase broth cultures of the isolates were transferred with the pin replicator to vitamin K1–enriched Brucella blood or chocolate (for H influenzae) agar plates and allowed to dry for 15 minutes at room temperature. A sample of a log-phase broth culture of the target strain was applied adjacent to each of the isolated strains, and the plates were incubated in 5% carbon dioxide or anaerobically at 37°C for 48 hours. Bacterial interference was considered present when inhibition of growth of the target strain was reproducibly detected adjacent to the isolated strains. Degrees of inhibition varied from complete absence of growth to a narrow zone of poor growth along the proximal area of the colony.
Statistical analysis was done using the Fisher exact test. The P values are 2 sided.
Eleven potential pathogens (0.92 per subject) were isolated from nasopharyngeal cultures obtained from 9 of the 20 individuals before smoking cessation, and 2 (0.17 per subject) were recovered from 2 individuals after smoking cessation (P<.05) (Table 1). One of these individuals was colonized with the same pathogen (H influenzae) prior to cessation of smoking.
Bacterial interference between 2 aerobic (α-streptococci and nonhemolytic streptococci) and 2 anaerobic species (Prevotella and Peptostreptococcus species) and 4 potential pathogens (S pneumoniae, H influenzae, M catarrhalis, and S pyogenes) was observed. Bacterial interference was noted in 35 instances against the 4 potential pathogens by 14 normal flora isolates that were recovered from the smokers before cessation, and in 116 instances by 42 isolates after cessation (P<.01) (Table 2).
This study compares the recovery rate of potential pathogens as well as interfering aerobic and anaerobic bacteria from the nasopharynges of smokers before cessation of smoking and 12 to 15 months after cessation of smoking. Our findings confirm those from our previous study9 that the nasopharyngeal flora of smokers contain fewer aerobic and anaerobic organisms with interfering capability and more potential pathogens compared with nonsmokers. However, to our knowledge, we have observed for the first time that the high number of pathogens and low number of interfering organisms revert to normal levels after complete cessation of smoking. These levels were similar to those we had observed in nonsmokers in a previous study.9
Haemophilus influenzae, S pneumoniae, and M catarrhalis were more frequently isolated in subjects who were prone to sinusitis12 and otitis media,13,14 and S pyogenes was more often isolated in children prone to tonsillitis15 than in those not prone to infections. Conversely, aerobic and anaerobic interfering organisms were more often recovered in those patients who were not prone to infection compared with those who were prone to upper respiratory tract infections.12-15 Smoking was a risk factor in patients with chronic obstructive pulmonary disease for colonization with Pseudomonas aeruginosa,16H influenzae,16,17S pneumoniae, and M catarrhalis.17
The presence of organisms with interfering potential may play a role in the prevention of upper respiratory tract infections. Their absence in smokers may contribute to their increased risk of acquiring respiratory pathogens and their greater susceptibility to respiratory infections.1 The enhanced binding of pathogenic bacteria to epithelial cells of smokers may account for their predominance.7 It is also possible that the prior frequent use of antibiotics in smokers may have reduced the number of organisms inhibitory to the growth of pathogens. However, we examined only individuals who had not received antibiotic drugs in the past 3 months; prior antibiotic use does not explain the lack of such organisms in these individuals.
The ability of the indigenous normal nasopharyngeal flora to inhibit colonization with potential pathogens has been reported in several studies of subjects with upper respiratory tract infections.18-22 α-Hemolytic streptococci were found to inhibit the colonization in patients and in vitro growth of a variety of pathogenic bacteria. These include S pneumoniae, S pyogenes, and S aureus.18-22 The production of bacteriocin and other inhibitory substances that suppress some bacterial growth, or utilization of nutrients in the nasopharyngeal environment essential for the potential pathogens, may explain this phenomenon.23
Therapeutic colonization of the nasopharynx with interfering bacteria was recently studied by Roos et al,24 who attempted to colonize children with recurrent tonsillitis with α-hemolytic streptococci or placebo. Clinical recurrences occurred in 1 (2%) of 51 children in the α-hemolytic streptococci group and 14 (23%) of 61 children in the placebo-treated group. Similarly, these investigators showed that recolonization with α-hemolytic streptococci with the ability to inhibit the growth of pathogens (“interfering” activity) reduced the recurrence of acute otitis media and the frequency of otitis media with effusion in susceptible children.25 Three months after colonization with α-hemolytic streptococci, 22 (42%) of the children given the streptococcal spray were free of otitis media and had a healthy tympanic membrane compared with 12 (22%) of those given placebo.
In conclusion, our study demonstrates the beneficial effects of smoking cessation in restoring the interfering aerobic and anaerobic bacteria, which are potentially beneficial bacteria that can interfere with the growth of potential pathogens. The possible role of interfering bacteria and the potential recolonization with α-hemolytic streptococci has yet to be studied in patients with chronic obstructive pulmonary disease who smoke. Further studies of smokers are warranted to investigate whether colonization of the nasopharynx with interfering organisms and/or cessation of smoking would be beneficial, allowing for the return of the normal inhibitory flora and the reduction in the number of pathogens.
Correspondence: Itzhak Brook, MD, MSc, 4431 Albemarle St NW, Washington, DC 20016 (IB6@georgetown.edu).
Submitted for Publication: June 1, 2006; final revision received September 9, 2006; accepted October 23, 2006.
Author Contributions: Drs Brook and Gober had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Brook. Acquisition of data: Brook and Gober. Analysis and interpretation of data: Brook. Drafting of the manuscript: Brook and Gober. Critical revision of the manuscript for important intellectual content: Brook. Administrative, technical, and material support: Brook and Gober. Study supervision: Brook.
Financial Disclosure: None reported.
1.Aronson
MDWiss
STBen
RLKomaroff
AL Association between cigarette smoking and acute respiratory tract illness in young adults.
JAMA 1982;248181- 183
PubMedGoogle ScholarCrossref 2.Musher
DMFainstein
V Adherence of
Staphylococcus aureus to pharyngeal cells from normal subjects, smokers, staphylococcal carriers and patients with viral infections.
In: Jeljaszewicz
J, ed.
Staphylococci and Staphylococcal Infections, Supplement 10. New York, NY:
Gustav Fischer Verlag; 1981:1011-1116
Google Scholar 3.Blackwell
CCWeir
DMJames
VS
et al. Secretor status, smoking and carriage of
Neisseria meningitidis. Epidemiol Infect 1990;104203- 209
PubMedGoogle ScholarCrossref 4.Blackwell
CCTzanakaki
GKremastinou
J
et al. Factors affecting carriage of
Neisseria meningitidis among Greek military recruits.
Epidemiol Infect 1992;108441- 448
PubMedGoogle ScholarCrossref 5.Stuart
JMCartwright
KADawson
JARickard
JNoah
ND Risk factors for meningococcal disease: a case control study in south west England.
Community Med 1988;10139- 146
Google Scholar 6.Pershagen
G Review of epidemiology in relation to passive smoking.
Arch Toxicol Suppl 1986;963- 73
PubMedGoogle Scholar 7.El Ahmer
OREssery
SDSaadi
AT
et al. The effect of cigarette smoke on adherence of respiratory pathogens to buccal epithelial cells.
FEMS Immunol Med Microbiol 1999;2327- 36
PubMedGoogle ScholarCrossref 8.Fujimori
IGoto
RKikushima
K
et al. Isolation of alpha-streptococci with inhibitory activity against pathogens, in the oral cavity and the effect of tobacco and gargling on oral flora.
Kansenshogaku Zasshi 1995;69133- 138
PubMedGoogle Scholar 9.Brook
IGober
AE Recovery of potential pathogens and interfering bacteria in the nasopharynx of smokers and nonsmokers.
Chest 2005;1272072- 2075
PubMedGoogle ScholarCrossref 10.Murray
PRBarron
EJPfaller
MATenover
FCYolken
RH Manual of Clinical Microbiology. 6th ed. Washington, DC: ASM Press; 1995
11.Grahn
EHolm
SEEkedahl
CRoos
K Interference of alpha-hemolytic streptococci isolated from tonsillar surface on beta-hemolytic streptococci (
Streptococcus pyogenes): a methodological study.
Zentralbl Bakteriol Mikrobiol Hyg A 1983;254459- 468
PubMedGoogle Scholar 12.Brook
IGober
AE Bacterial interference in the nasopharynx and nasal cavity of sinusitis prone and non-sinusitis prone children.
Acta Otolaryngol 1999;119832- 836
PubMedGoogle ScholarCrossref 13.Brook
IGober
AE In vitro bacterial interference in the nasopharynx of otitis media-prone and non–otitis media-prone children.
Arch Otolaryngol Head Neck Surg 2000;1261011- 1013
PubMedGoogle ScholarCrossref 14.Bernstein
JMSagahtaheri-Altaie
SDryjd
DMWactawski-Wende
J Bacterial interference in nasopharyngeal bacterial flora of otitis-prone and non-otitis-prone children.
Acta Otorhinolaryngol Belg 1994;481- 9
PubMedGoogle Scholar 15.Brook
IGober
AE Interference by aerobic and anaerobic bacteria in children with recurrent group A beta-hemolytic streptococcal tonsillitis.
Arch Otolaryngol Head Neck Surg 1999;125552- 554
PubMedGoogle ScholarCrossref 16.Miravitlles
MEspinosa
CFernandez-Laso
EMartos
JAMaldonado
JAGallego
MStudy Group of Bacterial Infection in COPD, Relationship between bacterial flora in sputum and functional impairment in patients with acute exacerbations of COPD.
Chest 1999;11640- 46
PubMedGoogle ScholarCrossref 17.Zalacain
RSobradillo
VAmilibia
J
et al. Predisposing factors to bacterial colonization in chronic obstructive pulmonary disease.
Eur Respir J 1999;13343- 348
PubMedGoogle ScholarCrossref 18.Crowe
CCSandera
WE
JrLongley
S Bacterial interference, II: role of the normal throat flora in prevention of colonization by group A
Streptococcus. J Infect Dis 1973;128527- 532
PubMedGoogle ScholarCrossref 19.Brook
IGober
AE Role of bacterial interference and beta-lactamase-producing bacteria in the failure of penicillin to eradicate group A streptococcal tonspharyngotonsillitis.
Arch Otolaryngol Head Neck Surg 1995;1211405- 1409
PubMedGoogle ScholarCrossref 20.Aly
RMaibach
HIShinefield
HRMandel
AStrauss
WG Bacterial interference among strains of
Staphylococcus aureus in man.
J Infect Dis 1974;129720- 724
PubMedGoogle ScholarCrossref 21.Johanson
WG
JrBlackstock
RPearce
AKSanford
JP The role of bacterial antagonism in pneumococcal colonization of the human pharynx.
J Lab Clin Med 1970;75946- 951
PubMedGoogle Scholar 22.Sanders
E Bacterial interference, I: its occurrence among the respiratory tract flora and characterization of inhibition of group A streptococci by viridans streptococci.
J Infect Dis 1969;120698- 707
PubMedGoogle ScholarCrossref 23.Patek
MHochmannova
JNesvera
JStransky
J Glutamicin CB II, a bacteriocin-like substance produced by
Corynebacterium glutamicum. Antonie Van Leeuwenhoek 1986;52129- 140
PubMedGoogle ScholarCrossref 24.Roos
KHolm
EGrahn-Hakansson
ELagergren
L Recolonization with selected α-streptococci for prophylaxis of recurrent streptococcal pharyngotonsillitis: a randomized placebo-controlled multicentre study.
Scand J Infect Dis 1996;28459- 462
PubMedGoogle ScholarCrossref 25.Roos
KHakansson
EGHolm
S Effect of recolonisation with “interfering” alpha streptococci on recurrences of acute and secretory otitis media in children: randomised placebo controlled trial.
BMJ 2001;322210- 212
PubMedGoogle ScholarCrossref