The Differential Effect of Cigarette Smoke on the Growth of Bacteria Found in Humans* Adam Ertel; Robert Eng, M.D.; and Sharon M. Smith, Ph.D.

The effect of cigarette smoke oa growth of those species of bacteria that are CODSidered common potential human pathogens was eumioed in mtro. Smoke from both mentholated and nonmentholated cigarettes inhibited the growth of Gram-positive cocci to a greater degree than that of Gram-negative rods. S~ au,..., Streptococcua pneumoniae, and a variety of other sbeptococci were inhibited at a smoke solution dilutioa of 1:8. Enteric bacteria

smokers, in addition to having a high C igarette predisposition to develop cancer of the respira-

tory and gastrointestinal epithelium, have tendencies to develop gingival infections, bronchitis, and pneumonia. These infections occur as a result of complex interaction of the host, bacteria, and the short- and long-term effects of cigarette smoke. There is a possibility that cigarette smoke may directly affect the normal flora of the mouth and thus permit a reselection of colonizing flora of species more resistant to smoke. The direct effects of smoke on the growth of many bacterial species isolated from humans are mostly unknown, although the effect of smoke on dental flora has been examined extensively. a-t It is important to determine these effects ofcigarette smoke on bacterial species and to determine the contribution of this reselection of the particular species in the pathogenesis of bacterial infections in smokers. MATERIALS AND METHODS

8tJcterlaliiDlatu Studies

All isolates studied were coUected from mouth cultures and respiratory tract cultures of patients OYer several months. The isolates were stored frozen at -70"C, and when needed, were thawed and grown on sheep blood tryptic soy agar or cbooolate agar (Hemophilus species) before testing. lftpartJtion of Smoke Solutfon

Smoke from two brands of cigarettes (Kool or Camel) was drawn through 10 ml of tryptic soy broth (BBL, Cockeysville, Md) to produce a minimum steady stream of bubbles. The smoke-saturated solution was &Iter sterilized by passing through a 0.22 p.M syringe •From the Infectious Disease Section, Medical Service (Mr. Ertel and Dr. Eng), Microbiology Section, Laboratory Service (Dr. Smith), Department of Veteraus AfFairs Medical Center, East of Medicine (Dr. Eng), DepartOrange, NJ, and the ~ent ment Of Laboratory Medicine and Pathology (Dr. SmithY, UMONJNew Jersey MediCal School, Newark. Manuscript received September 24; revision accepted February 15 Reprint reque&U: Dr. Eng. lnjectlmu DfMue (111), VA Medical Center, EMt Orange, NJ 07019

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such as Klebsiella, Enterobacter, and Pseudomonas were not afl'ected by a I: I dilution of the solution. As with the Gram-positive cocci, the Neisseria species and Braohamella were also inhibited at a dilutioa o£ 1:8. Culture results of the mouth of 15 smokers and 15 DODSmokers showed that the smokers have a propensity to develop heavy Gramnegative bacterial colcmizaticm. (Cheat 1991; 100:628-30)

&Iter (Gelman Sciences, Inc, Ann Arbor, Mich). Nicotine free-base (Sigma Chemical Co, St. Louis, Mo) solution was also prepared in tryptic soy broth by direct addition foUowed by &Iter sterilization. Growth lnhllntion 'lest

The smoke solutions from the two brands of cigarettes (Kool or Camel) and the nicotine solution were used in a two-fold microbroth dilution test4 to assess potency for inhibiting bacterial growth. The dilutions were performed in sterile 92-weU microliter plates using 50 p.L tryptic soy broth. A final inoculum of 5 X 10" cfu/ml of each species of bacteria was added to the columns of weDs. In the case of Streptococcw pneumonitle (pneumococci), 100 U/ml of catalase (Sigma Chemical Co) was incorporated into the dilution medium. In the case of Hemophilus, BranhameUa, and Neisseria, 10 percent Fildes enrichment (BBL) was incorporated into the dilution medium to ensure growth. Mouth Flora

Nonpatient volunteers had oropharynx cultures performed using culturettes (Culturette II, Marion Scienti&c, Kansas City, Mo~ The culturettes were applied to speci&c areas of the oral cavity to include the buccal mucosa, the upper surface of the tongue, and the bony and soft palates. The procedures for cultures were considered harmless and were exempt from informed human consent according to Health and Human Services Basic Policy for Protection of Human Research Subjects.• The culturettes were then submerged in 1 ml of tryptic soy broth and mixed vigorously by hand to produce a homogenous suspension of bacteria. Then a 0.1ml portion was spread onto tryptic soy agar plates containing 2.0 1glml of penicillin G (P6zer, Inc, New York). The penicillin was added to select for presence of Gram-negative bacilli. The agar plates were incubated overnight, the number of colony types was enumerated, and Gram-negative isolates were identi&ed (using API 20E, Analytab Products, Plain~ NY). RESULTS

With few exceptions, the Gram-positive cocci were more susceptible to growth inhibition by cigarette smoke than the Gram-negative bacilli (Fig 1). Pseudomonas aeruginosa, Escherichia coli, Klebsiella, Enterobacter, Serratia, Providencia, Citrobacter, and Acinetobacter were all resistant to a 1:1 dilution of the smoke solution. The Gram-positive cocci, with the

DISCUSSION

FrcuRE 1. The highest twofold dilution of the smoke solution (1/titer) that inhibited bacterial growth. Smoke from twu brands of cigarettes (Camel and Kool) yielded the same results. Gram-positive organisms are shown in the upper panel; and Gram-negative organisms, lower panel.

exception of Enterococcus faecalis, were susceptible to growth inhibition even at dilutions as high at 1:8. The three fastidious Gram-negative genera studied, Branhamella, Neisseria, and Hemophilus, were as susceptible to the e~ects of smoke as were the grampositive cocci. There may be little inhibitory differences between the smoke from a nonmentholated cigarette and a mentholated cigarette. Using the twofold dilution method, no differences were detected, and the results shown in the figures apply to both types of cigarette smoke. Nicotine also produced marked growth inhibition for the bacteria tested. The Gram-negative rods and Gram-positive cocci were inhibited by 16lwml. Staphylococcus aureus and E faecalis were more resistant to the effects of nicotine and were inhibited only at a higher concentration of32 f.LWml. Isolates of the three fastidious Gram-negative genera, Branhamella, Neisseria, and Hemophilus, were the most susceptible and were inhibited by as little as at 8 f.LWml of nicotine. Of the 15 smokers who had cultures performed, four had high densities of Gram-negative bacilli in the oral cavity (Thble 1). Three of the 15 nonsmokers also had Gram-negative bacilli isolated from in the mouth. However, bacterial densities were at 100-fold lower.

Tobacco has long been considered, especially in the premodern medical era, to have medicinal value. Tobacco and tobacco smoke have been used on infected body sites and have allegedly effected improvements if not cures. 7 Many of the components of cigarette smoke have been identified, and compounds such as nicotine and acrolein have been shown to be present in high concentrations. 8 The nicotine content in unfiltered cigarettes is in the range of 1 mg. 9 The smoke from this cigarette may transfer a variable portion of available nicotine to the 10 ml of culture broth used in these experiments, but a concentration as high as 1 mw10ml or 100 tJ.wml is potentially possible. Our results indicate that the bacterial species studied were susceptible to growth inhibition at much lower concentrations of nicotine. It is interesting to note that Branhamella and Neisseria were both more susceptible to nicotine inhibition than the other organisms, a conclusion similar to the findings using smoke solutions. Oralftora culture data from smokers tend to support the in vitro findings that smokers may have an overgrowth of the more smoke-resistant organisms. In the 15 smokers who had cultures, four had high colony counts of Gram-negative rods. Although Gram-negative rods were present in approximately the same percentage of patients who did not smoke, the densities of these Gram-negative bacilli were much lower in nonsmokers. Neither the in vitro results nor the results of culturing human oralftora can explain why Hemophilus influenzae and pneumococci are known to be the predominant pathogens in smokers when these two organisms are quite susceptible to inhibition by smoke. Two possible explanations may be offered. First, the site of colonization by these two organisms in chronic smokers is the trachea, which is usually sterile in nonsmokers. 10 Although a varying amount of Table

1-Summt~ry

Volunteer No. Nonsmokers Ito 13 14 15 Smokers 1 to 11 12

qfl'enicillin-lfetMiatat Mouth Flora in

Smolcera and Norumolrsn Bacterial Species

Density (cfu!Culture)*

No penicillin-resistant organisms Acinetobacter species Pseudomonas aerugino66

20 20

Klebsiells pneumonloe

30

No penicillin-resistant organisms Pseudomonas maltophllia

Citrobocter freundii 13 14 15

Enterobocter agglomerans Staphylococcus aureus Escherichia coli

10" 10" 10" 10" 10"

*The bacterial density in colony forming units per milliliter. CHEST I 100 I 3 I SEPTEMBER, 1891

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inhaled cigarette smoke penetrates into this area, the organisms may be embedded in a thick matrix of in8ammatory cells and tenacious secretions that may protect the organisms from the inhaled smoke. 11 Secondly, the tracheal epithelium in smokers is known to produce copious amounts of secretions of up to 0.6 Uday secondary to in8ammation and other abnormal stimulants. 11•13 This dilutional effect may then drastically reduce the smoke content of the fluids in the trachea to allow growth of smoke-sensitive species. The selection for these two species in the trachea must also involve other factors such as attachment, permissiveness of neutrophils for these two species, interaction of these organisms with compromised tracheal cilial action, 14 and mechanisms by these organisms to evade antibody-mediated immunity. We conclude that cigarette smoke has the potential to select Gram-negative bacilli in the oral cavity. This recolonization may contribute to the development of pneumonia during aspirations of large amounts of mouth contents. However, the mouth recolonization does not appear to contribute to alteration of the microflora in the trachea, which is more important10 in the pathogenesis of pneumonia during episodes of lapses of the pulmonary toiletry.

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6 7 8 9

10

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REFERENCES 1 Barden D. Viability of six species of normal oropharyngeal bacteria after exposure to cigarette smoke m oftro. Microbios

2

1981; 32:7-14 D, Smith E. An m vitro study of the exposure of mixed

Barde~

Sixth World Conference

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populations of normal oropharyngeal bacteria to cigarette smoke. Microbios 1979; 216:159-64 Bastiann RJ, Waite IM. EfFects of tobacco smoldug on plaque development and gingivitis. J Periodontol1978; 49:480-82 Colman G, Beigbton D, Cbalk AJ, WakeS. Cigarette smoking and the microbial Bora of the mouth. Aust Dent J 1976; 21:11118 Jones RN, Barry AL, Gavan TL, Washington JA D. Susceptibility tests: microdilution and macroclilution broth procedures. In: Lennette EH, Balows A, Hausler WJ Jr, Sbadomy HJ, eels. Manual of clinical microbiology. 4th ed. Washington, DC: American Society tor Microbiology; 1985; 101:972-77 Health and Human Services. Subpart A. Basic HHS policy tor protection of human subjects. Part X. Paragraph 46.117 C(2~ Federal Register 1981; 46:8366-92 Kyle RA, Sbampo MA. French diplomat proclaims the medicinal properties of tobacco. Mayo Clio Proc 1986; 61:758 Johnstone RAW, Plimmer JR. The cbemical constituents of tobacco and tobacco smoke. Chem Rev 19159; 59:885-936 Federal1iade Commission. Report on tar, nicotine, and carbon monoDde of the smoke of !00 variety of dprettes. WasiUngton, DC; 1981 Irwin RS, Erickson AD, Pratter, Corrao WM, Garrity FL, Meyers JR, et al. Prediction oftracbeobronchia colonization in current cigarette smolten with chronic obstructive bronchitis. J Infect Dis 1982; 145:234-41 Ryley HC, Brogant D. Variation in composition of sputum in chronic chest disase. Br J Exp Pathol1989; 49:625-33 Boyd EM, Ronan A. Excretion of respiJatmy tract ftuid. Am J Physiol1942; 135:43-51 Reid L. An experimental study of hypersecretion of mucus in the bronchial tree. Br J Pathol1963; 44:437-35 Raldeten N, Raldeten ML, Feldman D, Boykin MJ. Mammalian ciliated respirator epithelium: studies with particular reference to eft'ects of menthol, nicotine and smoke of mentholated and non-mentholated cigarettes. Arch Otolaryngol1952; 56:495-503

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The differential effect of cigarette smoke on the growth of bacteria found in humans.

The effect of cigarette smoke on growth of those species of bacteria that are considered common potential human pathogens was examined in vitro. Smoke...
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