AEROBIC AND ANAEROBIC BACTERIAL COUNTS OF NASAL WASHINGS:

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1 AEROBIC AND ANAEROBIC BACTERIAL COUNTS OF NASAL WASHINGS: PRESENCE OF ORGANISMS RESEMBLING CORYNEBACTERIUM ACNES ELINOR D. WATSON, NANCI J. HOFFMAN, RICHARD W. SIMMERS, AND THEODOR ROSEBURY Department of Bacteriology, School of Dentistry, Washington University, St. Louis, Missouri Received for publication July 20, 1961 ABSTRACT WATSON, ELINOR D. (Washington University, St. Louis, Mo.), NANCI J. HOFFMAN, RICHARD W. SIMMERS, AND THEODOR ROSEBURY. Aerobic and anaerobic bacterial counts of nasal washings: Presence of organisms resembling Corynebacterium acnes. J. Bacteriol. 83: Aerobic and anaerobic colony counts in blood agar were made of 40 samples of nasal washings from five healthy adults. The anaerobic counts were consistently and significantly higher, in line with findings previously reported by others for skin, saliva, and feces. Catalase-forming diphtheroids utilizing lactate anaerobically, tentatively identified as members of the Corynebacterium acnes group, were conspicuous in the anaerobic cultures. 144 Anaerobic cultures of material taken from the healthy nasal passages seem never to have been made. The earlier literature on the normal flora of this area was reviewed by Kiister (1929) and Thomson and Thomson (1932). More recent studies were reported by Jacobson and Dick (1941), 'Masters et al. (1958), Kortekangas (1959), Lees and MeNaught (1959), and Harvey and Dunlap (1960, 1961). Aerobic cultures of the mucous membrane posterior to the vrestibule have usually revealed a scanty flora, most commonly of white staphylococci and diptheroids. Such cultures have sometimes been sterile. Thomson and Thomson (1932) stated that "our own experience is that the healthy nose does not harbor any definite variety of bacterium permanently, such as occurs in the month and intestine." But it seems unlikely that so exposed an area should lack a normal flora. Carriage in the nose of pathogenic staphylocceci (Miles, Williams, and Clayton- Cooper, 1940), diphtheria bacilli (e.g., Russell, 1943), and hemolytic streptococei (Hamburger, Green, and Hamburger, 1945), as well as pneumococci and influenza bacilli (Harvey and Dunlap, 1961), suggests that the nose is not necessarily an unfavorable site for bacterial colonization. When it has been possible to compare colony counts of indigenous bacteria in a given location by aerobic and anaerobic methods, the anaerobic counts have usually been conspicuously higher and have included species not found aerobically. This has been true of skin (Evans et al., 1950), of saliva (Richardson and Jones, 1958), and especially of feces (Eggerth and Gagnon, 1933). An available method for counting bacteria from the nasal cavities (Grubb and Puetzer, 1947) provided a means to supply the missing information on anaerobes in the nose. MATERIALS AND METHODS Subjects were five members of the laboratory staff (3 women, 2 men) aged 22 to 56 years, with no apparent nasal disease. Irrigating devices, as described by Grubb and Puetzer (1947), were made of Pyrex glass. A separate sterile device was used for each nostril, which was washed three times successively in 20 ml of Ringer's solution containing 0.1% gelatin. The five subjects were tested each morning on four successive days. Although the irrigating device was designed to avoid contamination of the washings with the vestibular flora, a further effort to prevent such contamination was made, especially in view of the known presence of anaerobes on the skin of this region (Pachtman, Vicher, and Brunner, 1954). The vestibule was cleansed before irrigation, using for one nostril in all instances soap and water on a large cotton swab followed by sterile 0.85% NaCl, and for the other nostril, on two of the test days, sterile saline alone, and on the other two days, 1:10,000 Zephiran (Winthrop Laboratories, New York, N. Y.) followed as before by sterile saline. The washings were shaken in sterile stoppered bottles (Grubb and Puetzer, 1947), and 10-fold dilutions in Ringer-gelatin solution were plated in duplicate in 5% rabbit

2 1962] CORYNEBACTERIUM AGCNES IN NASAL WASHINGS 145 blood-blood agar base (Difco Laboratories, Detroit, Mich.). After recovery of anaerobes by this procedure, an additional check of the qualitative findings was made by obtaining swab samplings through a sterile speculum prepared as a 5-cm length of 13-mm (outside diameter) tubing. The swabs were streaked on duplicate plates of rabbit blood agar. The plates were incubated at 37 C, one set aerobically for 48 hr, the other anaerobically in Brewer jars with hydrogen and 5% CO2 for 8 days. The dilution plates were counted and the bacterial flora identified broadly by morphological means, except as noted. The finding on the anaerobic plates of many more diphtheroids than appeared aerobically prompted an attempt to determine whether any of them were Corynebacterium acnes. Tests were made by standard methods (Society of American Bacteriologists, 1957) and repeated by methods given by Douglas and Gunter (1946) and Gutierrez (1953). Lactate utilization was identified by growth through at least three consecutive anaerobic subcultures on a medium, containing only sodium lactate and yeast extract, devised for isolation of Veillolla (Douglas, 1950). The statistical data in Table 1 were calculated by means of formulas given by Bailey (1959) for comparing two Poisson distributions (no. 14) and for the correlation coefficient (no. 23) and its t test (no. 25). RESULTS Colony counts. Aerobic and anaerobic counts are grouped in Table 1 by the method used for cleansing the nasal vestibule. The first or saline group included two high values (4,000 and 8,220 colonies per ml of washings), both from a single subject; the third highest value in this group, 1,900, was below the upper limit for the other aerobic groups. These findings, strengthened by the qualitative similarity of the flora from washings and from swab cultures, made it seem probable that the vestibular flora was successfully avoided. Hence the values were pooled to obtain two groups of 40 samples each for the aerobic and anaerobic counts. Of the 40 pairs of counts, the anaerobic value was larger in 36, equal in 1, and smaller in 3, the last including the maximum value in the aerobic group, 8,220. A distinct tendency for either count to be high when the other was high is indicated by the correlation coefficient. This and the difference between the group means appear to be clearly significant (Table 1). Qualitative findings. On the aerobic plates, diphtheroids and white staphylococci were found in all five subjects, both in the washings and on plates streaked with swabs. Found less regularly were a yellow staphylococcus, nonhemolytic streptococci, and gram-negative bacilli (one of which was Klebsiella-like). The anaerobic plates prepared both from washings and by streaking with swabs revealed from all subjects diphtheroids that grew anaerobically but not aerobically in subculture on blood agar, as well as staphylococci or micrococci that grew aerobically in subculture and gram-positive rods that failed to grow either aerobically or anaerobically in subculture. Aerobic diphtheroids were recovered irregularly on the anaerobic plates, and in one instance a gram-negative rod appeared but failed to grow in subculture. Eight cultures of diphtheroids isolated from anaerobic plates, four from nasal washings, and four from swab plates, were tested as putative C TABLE 1. Aerobic and anaerobic colony counts of nasal washings on blood agar* Aerobic Anaerobic Vestibule treated with Number of Number of samples Range Mean samples Range Mean 0.85% NaCl Soap and 0.85% NaCl :10,000 Zephiran and 0.85% NaCl Total * Ranges and means expressed as: X 102 per ml of washings. Group means compared: d = 22.97, P < Correlation of aerobic-anaerobic paired data: r = +0.65, t = 6.94, P = <0.001.

3 146 WATSON ET AL. [VOL. 83 TABLE 2. Comparative findings for anaerobic diphtheroids isolated from the nosea vz.4.,0 a 6 a n E Organism 0E- a Nose cultures N1, N11, N14, Ab 40c + A A A Na2, NblO N A od + A A A Na A +4± + A A A Aw 0 0 Na Ar ++ + A A A A 0 A Douglas and Gunter (1946): C. acnes V ACp + +e A A A Gutierrez (1953): C. acnes + + V Cp + + A Av Av V Ov King and Meyer (1957): anaerobic diph- + V + ACr V A A 0 A V theroids C. acnes ACr 0 A A V A 0 a Symbols used: A, acid; r, reduced; C, clotted; p, peptonized; V, variable; v (A,, Ov), usually; w, weak. b Acidified and reduced by Nll and NblO in one test (S.A.B. method). c Weak liquefaction by one or both methods (S.A.B., Douglas and Gunter); strains Na2 and NblO did not grow on the S.A.B. gelatin medium. d No growth on S.A.B. gelatin medium; negative in Douglas-Gunter medium. e Gutierrez stated that Douglas and Gunter found lactate utilization after having reported it as negative in acnes. The findings are given in the upper part of Table 2. The six strains listed in the first two rows were uniform in their properties, except for reduction as well as acidification of litmus milk by two strains, and variation in gelatin liquefaction, which was weak or negative for all strains. The seventh strain, Na6, differed from the preceding six by showing well marked gelatin liquefaction in one of two test methods, and by weakly fermenting raffinose. The eighth strain, Na4, was distinctive among the group in being hemolytic on both sheep and rabbit blood agar plates, in failing to reduce nitrate, and in giving strong positive reactions in gelatin, raffinose, and lactose. In addition, strain Na4 showed yellowish colonies rather than the whitish colonies of the other strains, and grew aerobically on blood agar after 72 hr at 37 C, whereas the other seven grew only under anaerobic conditions. DISCUSSION The findings in the present study support those of earlier workers in indicating that the bacterial flora of healthy nasal passages is usually sparse. The aerobic counts reported here are also generally consistent with those obtained by Grubb and Puetzer (1947) with the same method for four of their five healthy subjects, among whom the counts during 10 days ranged from 70 to 13,000 (per ml of a 20-ml sample of washings), with a mean of 1,410. Corresponding values in this study were 30 to 8,220, with a mean of 1,050. The fifth subject studied by Grubb and Puetzer gave higher counts; the mean of their entire group was 4,330 per ml. The presence in the nasal passages of bacteria that grow preferably or exclusively under anaerobic conditions, and the finding that such organisms outnumber aerobes, may seem surprising in view of the ventilation of the area. A similar relationship holds, however, on skin (Evans et al., 1950), including an exposed area of the face (Pachtman et al., 1954). There is evidence (e.g., Lovell, 1945) that the indigenous or "resident" bacteria of skin, including the anaerobes, are

4 19621 CORYNEBACTERIUM ACNES IN NASAL WASHINGS 147 typically found deep in hair follicles and sebaceous glands, where access of oxygen would be limited; analogous circumstances may apply in the nasal passages. Anaerobes, moreover, can of course be grown in vitro without excluding oxygen if suitable reducing agents are present. Reducing conditions are presumably provided in vivo by aerobic bacteria and host tissue cells. No attempt was made in this study to identify all the anaerobic bacteria of the nose. The conspicuous anaerobes recovered were catalaseforming diphtheroids that resemble C. acnes. The comparative data in the lower part of Table 2 show that seven of the eight nasal diphtheroids agree with the C. acnes of Douglas and Gunter (1946) and Gutierrez (1953) in the formation of catalase, the reduction of nitrate, the anaerobic utilization of lactate, and generally, in their fermentation reactions. They differ, however, in being nonhemolytic, in failing to coagulate milk, and in being only weakly active or inactive in liquefaction of gelatin. Except for action on milk and indole formation, the nasal cultures agree with both the C. acnes and some of the "anaerobic diphtheroids" described by King and Meyer (1957); it is of interest that one of the C. acnes strains reported on by these workers (ATCC 6923) was also studied by Douglas and Gunter (1946). The eighth strain of the present study, Na4, failed both to reduce nitrate and to clot milk; but its other characteristics, including hemolysis and gelatin liquefaction, are similar to those of C. acnes. Some of the anaerobic diphtheroids of King and Meyer (1957) were also nitrate-negative. King and Meyer reported that 43 of 60 strains of anaerobic diphtheroids cross-agglutinated with antiserum prepared against C. acnes ATCC 6923; 42 of these strains agglutinated with another C. acnes antiserum. Cross-agglutination was also shown by five C. acnes cultures in two anaerobic diphtheroid sera but not in two others. Neither C. acnes nor the anaerobic diphtheroids cross-reacted with anaerobic actinomycetes or with the so-called Lactobacillus bifidus. Pending further study of these organisms the writers would tentatively include both the anaerobic diphtheroids of King and Meyer and those recovered in this study as members of the C. acnes group. ACKNOWLEDGMENT This work was aided by research grant E3113 from the National Institute of Allergy and Infectious Diseases, U. S. Public Health Service. LITERATURE CITED BAILEY, N. T. J Statistical methods in biology. John Wiley & Sons, New York. DOUGLAS, H. C On the occurrence of the lactate fermenting anaerobe, Micrococcus lactilyticus, in human saliva. J. Dental Research 29: DOUGLAS, H. C., AND S. E. GUNTER The taxonomic position of Corynebacterium acnes. J. Bacteriol. 52: EGGERTH, A. H., AND B. H. GAGNON The bacteroides of human feces. J. Bacteriol. 25: EVANS, C. A., W. M. SMITH, E. A. JOHNSTON, AND E. R. GIBLETT Bacterial flora of the normal human skin. J. Invest. Dermatol. 15: GRUBB, T. C., AND B. PUETZER A method for counting bacteria in the nasal cavity. J. Lab. Clin. Med. 32: GUTIERREZ, J Numbers and characteristics of lactate utilizing organisms in the rumen of cattle. J. Bacteriol. 66: HAMBURGER, M., JR., M. J. GREEN, AND V. G. HAMBURGER The problem of the "dangerous carrier" of hemolytic streptococci. I. Numbers of hemolytic streptococci expelled by carriers with positive and negative nose cultures. J. Infectious Diseases 77: HARVEY, H. S., AND M. B. DUNLAP Upper respiratory flora of husbands and wives. New Engl. J. Med. 262: HARVEY, H. S., AND M. B. DUNLAP Seasonal prevalence of upper respiratory pathogens. New Engl. J. Med. 264: JACOBSON, L. O., AND G. F. DICK Normal and abnormal bacterial flora of the nose. J. Am. Med. Assoc. 117: KING, S., AND E. MEYER Metabolic and serologic differentiation of Actinomyces bovis and "anaerobic diphtheroids. " J. Bacteriol. 74: KORTEKANGAS, A. E Investigations of the bacterial flora of the respiratory tract. Acta Oto-Laryngol., Suppl. 150: KtSTER, E Normale Bakterienflora in Mund, Nasenh6hle und Vagine bei Mensch und Tier II. Die Bakterien in der gesunden Nase. In W. Kolle, R. Kraus, and P. Uhlenhuth, [eds.], Kolle undwassermann Handbuch der pathogenen Mikroorganismen. Gustav Fischer, Jena, v. 6, p

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