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(Chest. 2005;127:2072-2075.)
© 2005 American College of Chest Physicians

Recovery of Potential Pathogens and Interfering Bacteria in the Nasopharynx of Smokers and Nonsmokers*

Itzhak Brook, MD, MSc and Alan E. Gober, MD

* From the Department of Pediatrics, Georgetown University School of Medicine, Washington, DC.

Correspondence to: Itzhak Brook, MD, MSc, 4431 Albemarle St NW, Washington, DC 20016; e-mail: ib6{at}georgetown.edu


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Active smoking and passive exposure to cigarette smoke are associated with colonization by some potentially pathogenic species of bacteria and an increased risk of respiratory tract infection in both adults and children. In an attempt to explain these observations, this study compared the frequency of isolation of potential pathogens, and aerobic and anaerobic bacteria that possess interfering capabilities (ie, interfering with the in vitro growth of potential pathogens) in the nasopharynx of smokers to their recovery in nonsmokers.

Methods: Nasopharyngeal specimens for cultures were taken from 20 smokers and 20 nonsmokers. Potential pathogens, and aerobic and anaerobic bacteria with interfering capabilities against these organisms were identified.

Results: Fourteen potential pathogens (0.7 per patient) were isolated from nasopharyngeal cultures obtained from 11 of the 20 smokers, and 4 (0.2 per patient) were recovered from 3 of the 20 nonsmokers (p < 0.01). In vitro bacterial interference between two aerobic ({alpha}-hemolytic and nonhemolytic streptococci) and two anaerobic species (Prevotella and Peptostreptococcus species), and four potential pathogens (Streptococcus pneumoniae, Haemophilus influenzae [non-type b], Moraxella catarrhalis, and Streptococcus pyogenes) was observed. Bacterial interference was noted in 61 instances against the four potential pathogens by 22 normal flora isolates that were recovered from the group of smokers, and in 155 instances by 50 isolates from the group of nonsmokers (p < 0.01).

Conclusions: These findings illustrate for the first time that the nasopharyngeal flora of smokers contains fewer aerobic and anaerobic organisms with interfering capabilities and more potential pathogens compared with those of nonsmokers.

Key Words: {alpha}-hemolytic streptococci • interference • Haemophilus influenzae • nasal flora • nonhemolytic streptococci • Peptostreptococcus sp • Prevotella sp • smoking • Streptococcus pneumoniae


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Smoking is associated with an increased risk of respiratory tract infection in adults1 and also with oral colonization by some potentially pathogenic microorganisms.234 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 the carriage of potentially pathogenic species of bacteria in both adults and children,6 possibly due to enhanced bacterial binding to the epithelial cells of smokers7 and the low number of {alpha}-hemolytic streptococci with inhibitory activity against Streptococcus pyogenes in the oral cavities of smokers.8 However, the rate of colonization with these as well as with other aerobic and anaerobic organisms with interfering capabilities has not been evaluated previously. The purpose of the study was to determine the effect of active smoking on the frequency of recovery of potential pathogens and aerobic and anaerobic interfering bacteria in the nasopharynx of smokers, and to compare their recovery to that in nonsmokers.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients
Forty healthy adults (20 smokers; defined as smoking at least 10 cigarettes a day for the past 5 years) were included in the study. None had any chronic illnesses, and none had received antimicrobial therapy or had a respiratory tract infection in the past 3 months. Patients’ ages varied from 21 to 48 years (average, 31 years), and 31 patients were men. Age and sex distributions were similar in both groups. The study was conducted between November 10, 2001, and March 21, 2002. Cultures were obtained using sterile calcium alginate swabs. Specimens were collected from the retropharynx (through the mouth), and were immediately plated into media that was supportive of the growth of aerobic and anaerobic bacteria.

Microbiology
Sheep 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 < 5% carbon dioxide (blood and chocolate agars), and they were examined at 24 h and 48 h. For the recovery of anaerobic bacteria, the specimens were inoculated onto prereduced vitamin K1-enriched Brucella blood agar, blood agar that contained kanamycin and vancomycin, and an aerobic blood plate that contained phenylethyl alcohol and enriched thioglycolate broth. These media were immediately incubated in jars (Gas Pack; Becton Dickinson; Franklin Lakes, NJ) at 37°C, and were examined after 48 h and 96 h of incubation at 37°C. All types of colonies on each plate were isolated. The total of aerobic and anaerobic bacterial isolates processed from each individual varied from 4 to 17 (average, 7.8 isolates). Aerobic and anaerobic bacteria were identified by previously described methods.910

Testing for Interference
The inhibitory activity was tested in a blind fashion, against one strain each of a recent clinical isolate of Streptococcus pneumoniae, Haemophilus influenzae (non-type b), Moraxella catarrhalis, and S pyogenes. The inhibitory activity of five 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 were allowed to dry for 15 min 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 h. Bacterial interference was considered to be present when the inhibition of the 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 significance was calculated using the {chi}2 test with Yates correction.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Fourteen potential pathogens (0.7 per patient) were isolated from nasopharyngeal cultures obtained from 11 of the 20 smokers, and 4 potential pathogens (0.2 per patient) were recovered from 3 of the 20 nonsmokers (p < 0.01) [Table 1 ].


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  		Table 1. Isolates of Pathogens Found in the Nasopharynx in 20 Smokers and 20 Nonsmokers*

 
Bacterial interference between two aerobic ({alpha}-hemolytic and nonhemolytic streptococci) and two anaerobic species (Prevotella and Peptostreptococcus species), and four potential pathogens (S pneumoniae, H influenzae, M catarrhalis, and S pyogenes) was observed. Bacterial interference was noted in 61 instances against the four potential pathogens by 22 normal flora isolates that were recovered from the smokers group, and in 155 instances by 50 isolates from the nonsmokers group (p < 0.01) [Table 2 ].


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  		Table 2. Isolates With Interfering Capabilities Recovered in the Nasopharynx in 20 Smokers and 20 Nonsmokers*

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The study compared the recovery rate of potential pathogens as well as interfering aerobic and anaerobic bacteria from the nasopharynx of individuals who smoked and did not smoke, and illustrated for the first time that the flora of smokers contains fewer aerobic and anaerobic organisms with interfering capability and more potential pathogens compared with those of nonsmokers. H influenzae, S pneumoniae, and M catarrhalis were more frequently isolated in patients prone to sinusitis12 and otitis media,1314 and S pyogenes was more often isolated in tonsillitis-prone children15 than in non-infection-prone patients. Conversely, aerobic and anaerobic interfering organisms were more often recovered in non-infection-prone patients compared to patients who are prone to upper respiratory tract infection (URTI).12131415 Smoking was a risk factor in patients with COPD for colonization with P aeruginosa,16 H influenzae,1617 S pneumoniae, and M catarrhalis.17

The presence of organisms with interfering potential may play a role in the prevention of URTIs. It is possible that the prior frequent use of antibiotics in infection-prone individuals may have reduced the number of organisms that are inhibitory to the growth of pathogens. However, since we studied only individuals who did had not received antibiotics in the previous 3 months, prior antibiotic use does not explain the bacterial discrepancies found between smokers and nonsmokers.

The ability of the indigenous normal nasopharyngeal flora to inhibit colonization with potential pathogens has been studied in several URTIs.1819202122 {alpha}-hemolytic streptococci were found to inhibit the colonization of a variety of pathogenic bacteria in patients and to inhibit them during in vitro growth. These bacteria include S pneumoniae, S pyogenes, and Staphylococcus aureus.1819202122 The production of bacteriocin and other inhibitory substances that suppress some bacterial growth, or the utilization of nutrients in the nasopharyngeal environment that are essential for the growth of potential pathogens, may explain this phenomenon.23

We were able to show for the first time in smokers that organisms other than {alpha}-hemolytic streptococci can also interfere with the in vitro growth of potential pathogens. These pathogens included aerobic nonhemolytic streptococci as well as the anaerobic bacteria species Peptostreptococcus and Prevotella.

Therapeutic colonization of the nasopharynx with interfering bacteria has been studied by Roos et al,24 who implanted either {alpha}-hemolytic streptococci or placebo in children with tonsillitis. Clinical recurrences occurred in 2% of patients in the {alpha}-hemolytic streptococci group (1 of 51 children) and in 23% of the placebo-treated group (14 of 61). Similarly, these investigators showed that recolonization with {alpha}-streptococci with the ability to inhibit the growth of pathogens (ie, the 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 {alpha}-streptococci, 22 of the children (42%) to whom the streptococcal spray had been administered were otitis media-free and had a normal tympanic membrane compared with 12 of those patients (22%) who had been given placebo.

The possible role of interfering bacteria and the potential recolonization with {alpha}-streptococci has yet to be studied in COPD patients who smoke. Further studies in smokers are warranted to investigate whether colonization of the nasopharynx with interfering organisms and/or the cessation of smoking would be beneficial, and whether they would allow for the return of the normal inhibitory flora and a reduction in the number of pathogens.


   Footnotes
 
Abbreviation: URTI = upper respiratory tract infection

Received for publication May 12, 2004. Accepted for publication November 11, 2004.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Aronson, MD, Wiss, ST, Ben, RL, et al (1982) Association between cigarette smoking and acute respiratory tract illness in young adults. JAMA 248,181-183[Abstract]
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I. Brook and A. E. Gober
Effect of Smoking Cessation on the Microbial Flora
Arch Otolaryngol Head Neck Surg, February 1, 2007; 133(2): 135 - 138.
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