Arch. oral Bid. Vol.10, pp.61-70, 1965. Pergamon Press Ltd. Printed in Cit. Britain

THE INDIGENOUS ORAL FLORA OF MAN-I

THE NEWBORN TO THE I-YEAR-OLD INFANT

CHARLOTTE MCCARTHY, M. L. SNYDER and R. B. PARKER

Department of Bacteriology,

University of Oregon Dental School, Portland, Oregon

Summary-Oral samples obtained from a group of fifty-one newborn and fortyfour- month-old infants and repeat specimens collected from the latter group at the ages of 8 and 12 months yielded a total of 153 oral specimens. The incidence of thirteen bacterial genera was determined. Only species of Streptococcus, Staphylococcus, Veilionella and Neisseria were constantly present by 12 months of age; Actinomyces, Lactobacillus, Nocardia and Fusobacterium species were cultured in more than half the subjects at this age, while Bacteroides. Leptotrichia, Candida, Corynebacterium species and the coliform types were isolated from less than half of these infants. Quan- titatively, the average total bacterial counts were of the order 104-5 per milligram of sample. Streptococci were dominant but the percentage diminished from 98 per cent to 70 per cent of the total by the end of the first year of life. The accretion of filamentous and branching forms was found to occur with advancing age but did not appear to be solely dependent upon tooth eruption since these forms could be isolated from pre- dentate infants.

INTRODUCTION

THE VARIOUS bacteria comprising the indigenous oral flora of man at birth and the

early months of life have been described by a number of investigators: LEWKOWICZ

(1901) JEANNIN (1904), BRWLOVSKY-LOUNKEVITCH (1915), KOSTECKA (1924), TORREY

and REESE (1945), HURST (1956), BERGER, KAPOVITS and PFEIFER (1959), ONISI, KOIKE

and TACHIBANA (1959), and others. There is general agreement that the mouth of

man is usually devoid of micro-organisms at birth but is soon contaminated with a flora primarily coccal in nature. However, the proposal by KOSTECKA (1924) that the

flora of the edentulous mouth is only aerobic is not supported by the earlier observa- tions of LEWKOWICZ (1901) who first described an anaerobic coccus Micrococcus gazogenes alcalescens anaerobius, in the edentulous mouths of infants. We recognize this organism today as VeilloneZZa akalescens. Subsequently HURST (1957), BERGER

et al. (1959), and ONISI et al. (1959) reported isolation of fusobacteria from the buccal cavities of infants who were two months of age and younger. KOSTECKA also specu-

lated that once established the oral species are retained and the appearance of new species is one of accretion and not of replacement.

On the other hand there is a paucity of information about relative numbers in respect to increasing age and dentition. We have attempted to map these changes by culturing identifiable genera listed in the papers of MORRIS (1953 a, b, 1954 ad) and RICHARDSON and JONES (1958) as well as in the text of BESET and DAVIS (1960).

61

62 CHARWTTE MCCARTHY, M. L. SNYDER AND R. B. PARKER

Our efforts have been limited to thirteen genera that could be selectively isolated without enrichment. The present paper reports the incidence and numbers of these organisms in the first year of life.

MATERIALS AND METHODS

Infants in the present study are subjects in a Child Development Study at the University of Oregon Medical School. These are randomly selected at birth and examined periodically then and at 4, 8, 12, 18 and 36 months of age.

A total of 153 oral samples were taken from two groups : one was limited to fifty- one newborn and the other to forty-four infants approximately 4 months of age. The latter group served as a source for additional specimens but only twenty-nine, and these were not always the same, were seen again at 8 and 12 months as they returned for periodic examination.

A sterile cotton swab inserted in the stopper of a disposable plastic tube or “swube” (Falcon Plastics) was used in obtaining each sample. The “swube” con- tained 2.5 ml of brain heart infusion broth with 0.1 per cent agar, and the entire assembly was weighed on a Mettler H-5 balance. Specimens were obtained by rubbing the dry cotton swab over the upper and lower dental ridges and the teeth if present. After immersion in the broth, the increase in weight of the “swube” was determined. The average wet weight of the oral material collected was about 100 mg. Control “swubes” were included for determination of loss of weight due to evapora- tion of broth.

Within l-2 hr after collection the content of the swab was emulsified by mild agitation and pressure against the side of the tube. Tenfold dilutions of this suspension were made in 4.5 ml of plain brain heart infusion broth; a separate pipette was employed for each dilution. One-tenth millimeter from the original broth or appro- priate higher dilutions was spread over the surface of each of the various plates of selective media. Plates of anaerobic incubation were placed in pressure cookers adapted for anaerobic growth. These were evacuated and flushed three times with 90 per cent hydrogen and 10 per cent carbon dioxide; after the final flushing electrical heating units in the cookers were connected to the line for 15 min to ignite any residual oxygen. Both aerobic and anaerobic plates were incubated at 37°C for 4 days at which time the colonies on the plates were counted. The quantity of oral material in each dilution was determined and from this the number of organisms per milligram of oral sample calculated. The identification of genus was based on observation of colony types appearing on the selective media and confirmed by gram stain following the classification scheme of BREED, MURRAY and SMITH (1957). The only exception

was the genus Leptotrichia which is not listed in this edition. The organisms sought and the selective media used for their isolation are listed as follows :

(1) Total aerobes and anaerobes: blood agar base (Difco) plus 5 per cent de- fibrinated sheep blood.

(2) Total streptococci: crystal violet azide agar (KRALJS and GASTON, 1956).

Anaerobic incubation.

THF. INDIGENOUS ORAL FLORA OF MAN-l 63

(3) Streptococcus salivarius: 5 per cent sucrose agar (SHERMAN, NIVEN and SMILEY, 1943) with sodium azide omitted. Aerobic incubation.

(4) Luctobacillus: Rogosa S L Medium (Difco) with 2400 units of nystatin per milliliter medium to inhibit growth of yeast. Anaerobic and aerobic incuba- tion.

(5) Veillonella: Rogosa Veillonella Medium (Difco) plus 7.5 pg vancomycin per milliliter medium (ROGOSA et al., 19.58). Anaerobic incubation.

(6) Neisseria: Chocolate blood agar containing 7.5 pg vancomycin per milliliter medium; addition of this antibiotic for selection of these organisms was our idea which proved helpful in elimination of gram positive organisms capable of giving a positive oxidase reaction. After incubation the inoculated plates were flooded with 1 per cent aqueous solution of N,N-dimethyl-p-phenylene- diamine monodrochloride. Colonies which turned pink and eventually black were counted as Neisseria (GORDON and MCLEOD, 1928). Aerobic incubation. It was found, however, that gram negative diplococci giving a positive oxidase reaction could also be cultured anaerobically.

(7) Staphylococcus: 5 per cent blood agar containing 8 per cent sodium chloride. Organisms from the oral cavity that grew on this medium were literally salt- tolerant micrococci; however, most of these were members of the genus Staphylococcus (CHAPMAN, 1945). Aerobic incubation.

(8) Bacteroides: 5 per cent blood agar with 200 pg of vancomycin per milliliter of medium (FINEGOLD, SIEWERT and HEWITT, 1957). Gram-negative rods which produced small usually colourless colonies and which could not be subcultured aerobically were counted as Bacteroides sp. Anaerobic incubation.

(9) Leptotrichia and Fusobacterium: the basic medium of BAIRD-PARKER (1957) with the addition of 5 pg crystal violet and 20 pg streptomycin per milliliter. Organisms were placed in the genus Leptotrichia if they resembled the descrip- tion of the genus proposed by GILMOUR, HOWELL and BIBBY (1961). Anaerobic incubation.

(10) Actinomyces and Nocardia: blood agar containing starch and basic fuchsin (HOWELL et aE., 1959). This medium was only semi-selective; close scrutiny of the colonies and frequent gram stains were required for identification of the members of the two genera. Aerobic and anaerobic incubation. Gram- positive filamentous branching organisms observed on the aerobic plates were counted as Nocardia and those observed on the anaerobic plates were counted as Actinomyces. Presumably some strains could grow under both condition< and would be included in both categories.

(11) Corynebacterium: Mueller tellurite agar (Difco). Gram-positive pleomorphic rods derived from black or grey colonies were counted as members of this genus. Gram-positive anaerobic diphtheroids observed to grow on blood agar but not on the tellurite medium were not included in the Corynebacterium tabulation. Aerobic incubation.

(12) Candida: Sabouraud dextrose agar (Difco) with 20 units of penicillin and 40 pg of streptomycin per milliliter medium. Aerobic incubation.

E

64 CHARLOTIE MCCARTHY, M. L. SNYDER AND R. B. PARKER

(13) Coliforms : eosin methylene blue agar (Difco). Aerobic incubation. (14) Enterococci: SF medium (Difco). One-tenth milliliter of the original dilution

of the oral samples were added to 5 ml of the medium and incubated at 45*5”C. Formation of yellow colour was an indication of the presence of enterococci. Aerobic incubation.

Detailed discussions and references concerning the dehydrated selective media are cited in the Difco Manual (1953). An average of thirty plates was inoculated from the various dilutions of each sample. Duplicate plates were not employed since most of the organisms grew on at least two of the media and thus provided corroborative counts. None of the media was completely selective and it was necessary to perform repeated gram stains to verify the presence and quantities of the various genera sought.

RESULTS

As expected, there was a wide range in the actual numbers of organisms in each of the different genera. The ranges for each genus and age period are listed in Table 1.

There was usually a striking difference between the actual numbers of organisms identified and the total viable count recorded which would suggest (1) that the sensitivity of the selective media employed was not particularly good, or (2) that these thirteen genera do not comprise the entire oral micropopulace. On a numerical basis the streptococci were the only organisms cultured consistently and in quantity which

TABLE 1. INCIDENCE AND RANGESOFNUMBERS OF THEDIFFERENT GENERA PERMILLIGRAM ORAL

SAMPLEISOLATEDFROMJNFANTSATFOURAGE LEVELSDURING THE FIRSTYEAROF LIFE

Mean age in days

1.8 (51) 101 (44) 248 (29) 365 (29) NO. NO. NO. NO. Pas. Range POS. Range POS. Range POS. Range

Aerobic Total Streptococcus salivarius Staphylococcus Neisseria Nocardia Lactobacillus, &x,,1;,,1,,

C&forms

Anaerobic Total Streptococcus Lactobacillus Actinomyces Veillonella Fusobacterium Bacteroides Leptotricbia

42 0 E-1,600,000

26 1.1-l 10.000 23 0.2-270

4 1.4-1.200 0 None 0 None 0 None 3 1.3-6 4 l-630,000

42 3-3,600,OOO 38 2-l,lOO,oOO

9 l-14.000 0 No& 1 390 0 None 0 None 0 None

44 1 l,OOO-1.900,000

33 lo-39,000 42 0.2-520 30 Z-3,600

1 540 6 0.8-54 6 0.7-5

18 0.2-590 13 0.2-680

44 14,OOtL2,400.OOO 44 2,200-860.000

‘f w” 3; ~~l;,loo

: EZ

29 12,000-2,400,ooo 29 15,000-690,000

28 l.l-68,000 2‘~ 3 lo-84,000 29 0.7-1,900 29 0.5-6.300 29 1~3-54,ooo 29 &Sl,OOO ;“o 100~~8”;p00 18 0.5-40,OOO

14 0.‘&310 14 0.2-910 10 0.2-600

7 0.2-160 14 0.2-51 13 0.3-81.000 6 0.7-11

29 24,000-1,600,OOO 29 17,000-1,300,OOO 16 0.2-30.000 6 12-3.600

24 0.241,OOO 8 0+15,000 3 49-580

15 0.2-5.9

29 l&000-l,OOO,OOO 29 7,000-690,000 15 0.2-7,700 17 4&27,000 29 34-40.000 is o.z-7d.ooo 13 22-2,700 12 0.1-80

ranged on the average from 98 per cent of culturable bacteria in the first day or two of life to 70 per cent at the end of the year. Since the total culturable counts did not change significantly over this period it would appear that the replacement of strepto- cocci occurred by substitution of other bacteria rather than by accretion as suggested by KOSTECKA. All genera sought were present by the fourth month in the oral flora

THE INDIGENOUS ORAL FLORA OF MAN-I 65

of these infants, even if only occasionally and in low numbers. A marked increase in frequency had occurred for members of several genera by the end of the first year.

At best these figures are relative in nature. Their changing relationships are more readily apparent when plotted graphically (Figs 1 and 2), for which purpose the

0 248 365

Mean age, days

FIG. 1. Graphic tabulation of the number of positive samples and the quantitative relationships of organisms cultured from them through aerobic incubation.

arithmetic figures were converted to logarithms (base 10). The mean of these logarithms for the various genera within each age group was taken as the most repre- sentative figure for that age group. A similar procedure is used in the dairy industry to give fair counts in milk aliquots (Milk Industry Foundation, 1959). The frequency of the incidence and the mean logarithm of the positive specimens for the different aerobic and anaerobic bacteria cultured are shown in Figures 1 and 2. This method of presentation not only illustrates frequency of occurrence of each genus at each level but also the relationship between the quantities and kinds of microorganisms that inhabit the infant mouth:

(a) Newborns Samples from nine of the newborn infants less than 24-hrs-old showed no growth

on any of the plates inoculated. As previously recorded by BRAILOVSKY-LOUNKEVITCH

(1915), we found the dominant genus present in the mouths of newborn children to be Streptococcus and the species Streptococcus salivarius in particular was isolated from one-half of the group. The genus Staphylococcus occurred in very low numbers

66 CHARLOTTE MCCARTHY, M. L. SNYDER AND R. B. PARKER

Lactobacillus ?

IO. positive sa lo. sompl

Age I.8 101

%%

-&R

to EJ 241 -

29 29

24

-55

Mean age, doys

FIG. 2. Graphic tabulation of the number of positive samples and the quantitative relationships of organisms cultured from them through anaerobic incubation.

also in about half of the newborns. These two genera could perhaps be considered indigenous to the mouth of the newborn infant. Strict anaerobes were limited to the anaerobic types of Lactobacillw and one strain of Veillonella.

(b) Other infants: The second group of forty-four infants showed a general increase in both variety

and numbers of organisms as they were sampled at 4, 8 and 12 months. Streptococci were numerically predominant (100,000 per milligram) and were found in all of the samples; S. salivarius was cultured from three-fourths of the 4-month-old and in all of the 12-month-old infants with counts that ranged from 1000 to 10,000 per milli- gram oral material. With the exception of two infants of 4 months, species of the genus Staphylococcus were invariably observed but on the average only in quantities of sixteen to forty viable cells per milligram of material. Gram-negative cocci of the genera Veillonella and Neisseria were constantly present in moderate numbers at the twelve month age level. Counts of Nocardia, Actinomyces, Bacteroides, Fusobacterium and Leptotrichia species ranged from moderately high to ve1.y low in 40-60 per cent of the subjects at 1 year of age. Lactobacillus species were piesent at all age levels; these were cultured in about half of the older infants. That anaerobic types were present was indicated by higher anaerobic than aerobic counts. Corynebacterium, Candida and the coliforms were observed only sporadically and in low numbers.

THEINDIGENOUSORAL FLORA OF MAN-I 61

Enterococci were determined to be present qualitatively in three of the 4-month-old infants, four of the %month-old infants and one of the 12-month-old infants.

The number of subjects was unfortunately too small to provide conclusive evidence about relationships between eruption of teeth and alteration of the oral flora. In this study teeth were occasionally seen in infants at 4 months of age, those of 8 months of age had a mean of two teeth (range O-8 teeth) and the 12-month-old infants had a mean of five teeth (O-12 teeth). However, insofar as the branching or filamentous forms commonly associated with plaques in tooth surfaces are concerned, it is interesting to compare the frequency of these forms isolated from pre-dentate and dentate children at 8 and 12 months respectively. The results are given in Table 2.

TABLE 2. THE INCJDENCEOF HIGHER MEMBERSOF THEORALFMRAINTHE

MOUTHSOFPRE-DENTATEANDDENTATECHILDRENFROMTHE8 AND 12 MONTH AGEGROUPS

Number samples from children

Organism

Bacteroides Fusobacterium Leptotrichia Nocardia Actinomyces Corynebacterium

13 Pre-dentate* 45 Dentatet Number Percent Number Percent positive positive positive positive

1 7.7 16 35.6 3 23.1 21 46’7 7 53.8 20 44.5 6 46.2 25 55.6 1 I.7 22 48.9 8 61.5 16 35.6

* Of the thirteen pre-dentate children, eleven were S-months and two were 13-months-old.

t Of the forty-five dentate children, eighteen were I-months-old and twenty-seven were 2-months-old.

Here it is noted that all of these organisms were isolated at one time or another from the edentulous of these older infants and only with Fusobacterium and Actino- myces was there an apparently significant increase in frequency of isolation as teeth erupt. This would indicate that factors other than teeth were involved in the establish- ment of Nocardia and Leptotrichia, but no clues about these could be discerned from the data obtained by this type of sampling.

DISCUSSION

It is difficult to determine the indigenous flora within an individual at a single moment in his life or even at various times in his life. This is particularly true of the infant at which time microbial changes are most striking. The moment an infant begins to feed a variety of microbes are introduced into his mouth. These are primarily transients but some find an environment suitable to their metabolism. Some organisms establish residence in certain individuals only, whereas others are a part of the oral flora in a large percentage of all humans as exemplified by the streptococci. Our

68 CHARLOTTE MCCARTHY, M. I-. SNYDER AND R. B. PARKER

qualitative data agree in most respects with similar studies by BRKILNSKY-LOUNKE- VITCH (1915), TORREY and REESE (1945), HURST (1956) and JORESS, COHEN and KREIDBERG (1960) on the nature of the oral flora in the infant.

In addition we attempted to determine the numbers of the different genera con- sidered normal members of the oral flora as they establish themselves along the dental ridge and teeth at the age levels sampled. Here we found, as ROSEBURY (1962) com- mented, that the total number of organisms cultured for all the genera observed does not equal the total viable counts recorded for the same material cultured on a non-selective medium. This means either that there were other organisms present in relatively large numbers not classifiable by the parameters employed or that the sensitivity of the selective media was low, requiring many cells of each microbial type before growth was observed. With increasing age, there was a definite influx of non-sporulating anaerobes and filamentous forms associated with a corresponding decline in the incidence of streptococci from 98 per cent of the total shortly after birth to approximately 70 per cent at 1 year of age when the full complement of the adult oral microflora was present in a majority of these infants. Unfortunately, the 4 months lapsing between samplings effectively prevented precise measurement of any change in the oral flora associated with the developing dentition.

Acknowledgement-This investigation was supported in part by grant DE 01423 from the United States Public Health Service.

R&&--Des tihantillons buccaux obtenus d’un groupe de 51 nouveau-n&s et d’enfants Ages de 44 mois et des sp&imens receuillis dans ce demier groupe aux Lges de 8 et 12 mois ont permis d’isoler 153 micro-organismes buccaux. Le frequence de 13 souches bactkriennes est d&ermin&. Seuls des streptocoques, staphylocoques, VeilloneNa et Neisserai sont constamment trouvts g 1’Lge d’un an: des esp&ces Actinomyces, Lacto- bacillus, Nocardia et Fusobacterium sont cultiv&s chez plus de la moitie des sujets de cet Lge, alors que des Bacteroides, Leptotrichia, Candida, Corynebacterium et des batteries colifonnes sont isol& dans moins de la moitie des enfants. Quantitative- ment, le nombre total moyen de bact&ies est de l’ordre de 104m5 par milligramme d’&hantillon. Les streptocoques sont predominants, mais le pourcentage diminue de 98 % & 70% du total B la fin de la premibre am& de la vie. La prtsence de formes filamenteuses et ramifiCes semble en rapport avec l’lge plus avanti mais ne parait pas like exclusivement g l’&uption dentaire, 6tant donnC que ces formes on pu &re isolQs chez des enfants avant l’&uption des dents.

Zusammenfassung-Mundhiihlenabstriche von einer Gruppe von 51 Neugeborenen sowie von 44 einen Monat alten Kindem und wiederholte, bei letzterer Gruppe im Alter von 8 und 12 Monaten gesammelte Proben umfassten insgesamt 153 Versuchs- muster. Dabei wurde das Auftreten von 13 Bakterienarten bestimmt. Bis zum Alter von 12 Monaten waren nur Arten von Streptococcus, Staphylococcus, Veillonella und Neisseria regelmbsig vorhanden. In mehr als der H&We der Fllle dieser Altersgruppen konnten Actinomyces-, LactobacilIus-, Nocardia- und Fusobacterium-Arten kulturell nachgewiesen werden. Species von Bacteroides, Leptotrichea, Candida, Coryne- bacterium und die Coli-lhnlichen Typen wurden dagegen von weniger als der Hllfte diese.r Kinder isoliert. In quantitativer Hinsicht lag der Durchschnitt aller Bakterien- zghlungen in der Grtissenordnung von lO*-6 pro mg der Probe. Streptococcen standen im Vordergrund, aber ihr prozentualer Anteil am Gesamtbefund verminderte sich mit dem Ende des ersten Lebensjahres von 98 auf 70%. Der Zuwachs filamentaser

THEINDIOENOZiSORAL FLORAOFMAN-I

und verzweigter Formen wurde mit fortschreitendem Alter beobachtet, aber er schien nicht allein vom Zahndurchbruch abhsngig zu sein, da diese Formen such von Kindem vor der Zahnung isoliert werden konnten.

69

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