Pediatric Pulmonology 44:497–502 (2009)

Oropharyngeal Flora in Healthy Infants:Observations and Implications for Cystic Fibrosis Care

David Carlson, MD,1 Elizabeth McKeen, MD,1 Michael Mitchell, MD,2 Brenda Torres, MS,2

Richard Parad, MD,3,4 Anne Marie Comeau, PhD,3 and Brian P. O’Sullivan, MD1*

Summary. Objective: The purpose of this preliminary study was to determine normal

oropharyngeal flora in healthy, non-CF infants in order to help care givers better interpret culture

results obtained from infants with CF. Methods: Oropharyngeal cultures were obtained from

104 healthy infants <12 months old. Cultures were obtained using the same methods as for CF

patients and were inoculated onto routine CF culture media. Approximately 20 infants from each of

5 age groups (0–2 days, 3 days to <3 months, 3 months to <6 months, 6 months to <9 months or

9 months to <1 year) were included in the well child sample. In addition, we reviewed serial results

of upper airway cultures obtained during the first year of life from 20 CF-affected infants whose

diagnosis was suggested by newborn screening. Results: Well infants in the first 48 hr of life had

very few pathogenic organisms found in their oropharyngeal cultures; 1/21 had S. aureus. Of the 83

samples from infants over 48 hr of age, we found that 27% (23/83) had S. aureus in their

oropharyngeal cultures. Many infants had polymicrobial cultures. Eleven percent of

culture samples had E. coli, E. cloacae, H. influenzae, or M. catarrhalis. Three of

83 cultures were positive for non-mucoid Ps. aeruginosa (3.6%), while 2 others were positive for

Ps. putida. Conclusion: Healthy infants can have multiple gram-negative and gram-

positive organisms recovered from their oropharynx. S. aureus and enteric gram-negative

organisms, including non-mucoid Ps. aeruginosa, can be found in the oropharynx of well children

up to 1 year of age. Care should be taken to not over interpret the presence of some of these

organisms in the oropharyngeal cultures of asymptomatic CF infants. Pediatr Pulmonol. 2009;

44:497–502. � 2009 Wiley-Liss, Inc.

Key words: cystic fibrosis; microbiology; throat culture; oropharynx; newborn

screening.

INTRODUCTION

The United States Cystic Fibrosis Foundation supportsnewborn screening for cystic fibrosis (CF) nationwide1

and studies in the U.S and United Kingdom have shownthat such screening is both medically warranted and costeffective.2–4 Newborn screening for CF is beneficial inpart because it allows for close monitoring of childrenbefore disease symptoms occur. The Cystic FibrosisFoundation recommends obtaining sputum or orophary-ngeal cultures at least quarterly as part of routinemonitoring for potential respiratory pathogens. In regionsperforming newborn screening, this means that physiciansare now confronted with the challenge of makingtherapeutic decisions based on culture results from pre-symptomatic infants. This problem is compounded whenthe infant identified as being at risk for CF via newbornscreening turns out to have an intermediate (borderline)sweat chloride test and genetic variants that may or maynot be disease causing. Results of throat culture swabsfrom these infants cannot be interpreted without knowingthe normal oropharyngeal flora from non-CF infants.

This information was presented in part at the 21st Annual North American

Cystic Fibrosis Conference, Anaheim, CA, October 2007.

1Department of Pediatrics, University of Massachusetts Medical School

and UMass Memorial Health Care, Worcester, Massachusetts.

2Department of Laboratory Medicine, University of Massachusetts Medical

School and UMass Memorial Health Care, Worcester, Massachusetts.

3New England Newborn Screening Program, University of Massachusetts

Medical School, Jamaica Plain, Massachusetts.

4Department of Newborn Medicine, Brigham and Women’s Hospital and

Harvard Medical School, Boston, Massachusetts.

*Correspondence to: Brian P. O’Sullivan, MD, 55 Lake Avenue, North;

Worcester, MA 01655. E-mail: osullivb@ummhc.org

Received 14 October 2008; Revised 10 December 2008; Accepted

14 December 2008.

DOI 10.1002/ppul.21029

Published online 9 April 2009 in Wiley InterScience

(www.interscience.wiley.com).

� 2009 Wiley-Liss, Inc.

Previous surveys of commensal bacteria in the oralcavities of infants have suffered from being skewedtoward detecting a small number of bacterial species,5 useof limited panels of DNA probes,6,7 being restricted toinfants under 10 days of age,8 or focusing on infantsin neonatal intensive care units.9,10 The results of thesestudies have not informed clinicians about the normalairway flora in healthy 1- to 12-month-old infants. Thus,inferring the clinical significance of the presence of aparticular bacterium in an oropharyngeal culture taken, forexample, from an apparently healthy 4-month-old infantwith CF is difficult; yet, interpretation of these data mayhave serious implications for treatment and/or isolationpractices.

This study was undertaken specifically to elucidatenormal oropharyngeal flora in infants under 1 year ofage who do not have CF. This information may allowphysicians caring for CF-affected infants to better under-stand the significance of culture results from their patients.

MATERIALS AND METHODS

Study Subjects

Infants between birth and 1 year of age were enrolled inthis study, with a goal of at least 20 subjects in each of fiveage ranges: 0–2 days, 3 days to <3 months, 3 months to<6 months, 6 months to <9 months, 9 months to <1 year.Full-term children 0–2 days of age were recruited fromthe well child newborn nursery at the UMass MemorialCampus; due to the fact that they had not yet had exposureto anything other than maternal vaginal flora their resultsare reported separately. Non-CF affected infants over2 days of age being seen for well-child care were enrolledin two settings, a hospital-based general pediatric out-patient clinic and a pediatric private practice in the city ofWorcester, MA. Exclusion criteria included a first-degreerelative with CF, respiratory illness at the time of culture,recent antibiotic exposure, or positive newborn screen forCF. Prior to obtaining the oropharyngeal culture, subjects’parents were asked to fill out a questionnaire pertaining toenvironmental exposures. The study was approved by theCommittee for the Protection of Human Subjects inResearch. Parents were given a fact sheet and asked toprovide verbal consent for the culture procedure.

Microbiology

Culture of the oropharynx was performed by using acotton-tipped swab and swabbing the posterior orophary-ngeal wall and tonsillar pillars. Samples were then placedin charcoal media and immediately transported to themicrobiology laboratory. Swab material was placed onstandard CF culture media consisting of blood agar plate,MacConkey agar plate, chocolate agar plate, OF PBL(oxidate fermentative basal media containing polymyxin,

bacitracin, and lactose), and BCG (bromo-cresolgreen) agar. All media were incubated at 358C. Allcultures were read once per day for 5 days or untilfinalized. The study samples were screened for thepresence of organisms considered to be significantcauses of lower respiratory tract infections in patientswith CF and other gram-negatives including: Beta-hemolytic Streptococci, Staphylococcus aureus, Strepto-coccus pneumoniae, Pseudomonas species, Burkhoderiacepacia, Haemophilus influenzae, M. catarrhalis, Enter-obacteriaceae, Escherichia coli, and yeast. Any growthof these organisms was considered significant. Alpha-hemolytic streptococci, non-pathogenic Neisseria spe-cies, Aerococcus viridians, and non-aureus staphylococciwere reported as mixed gram-positive and gram-negativegrowth.

Cystic Fibrosis Patients

Results of oropharyngeal cultures from CF patients wereobtained in a retrospective manner from chart review of all20 infants seen at the UMass CF Center from February 1999to July 2007 whose diagnosis was prompted by newbornscreening. Six of these children had genotypes consistentwith mild disease (three, F508del/R117H-7T; two,F508del/L206W; one, F508del/D1154N). Microbiologyrecords were reviewed for all oropharyngeal cultures takenfrom these infants from birth to 1 year of life. Culturesperformed at the time of clinic visits were obtained andprocessed in a manner identical to that described for thestudy subjects. Data pertaining to tobacco smoke exposureand breast-feeding were also extracted from the charts.

RESULTS

Non-CF Subjects

Oropharyngeal culture samples were obtained from 104healthy infants, ranging in age from birth to 1 year. Resultsof these cultures, divided by age groups, are shown inTable 1. Many subjects had more than one bacterialspecies recovered from the culture swab. Of note, only 1 of21 samples from patients in the first 48 hr of lifewas positive for an organism of interest (S. aureus).Seventeen of the 21 did have non-specific mixed gram-negative and gram-positive growth. The most commonlyseen organisms of interest in non-CF infants ages 3 daysthrough 1 year (n¼ 83) were S. aureus (27.7%) andCandida species (15.6%). E. coli, E. cloacae, H.influenzae, and M. catarrhalis, were each isolated with afrequency of �11%.

Three samples from non-CF patients grew non-mucoidPseudomonas aeruginosa; these were from a 3-month-oldchild and 10-month-old twin siblings. None of the threewas treated for this. Follow-up culture on the 3-month-old2 months later (5 months of age) demonstrated persistence

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498 Carlson et al.

of Ps. aeruginosa in the oropharynx. Repeat culture of thetwin siblings 5 months after the initial culture showedthat both had cleared the Ps. aeruginosa, but one hadPs. putida recovered from the subsequent culture at15 months of age.

Effects of tobacco smoke, breast-feeding, localenvironment, and recent antibiotic exposures wereanalyzed for their effect on amount and type oforopharyngeal colonization. No trends or statisticallysignificant effects were noted (date not shown).

CF Patients

Seventy-five oropharyngeal samples were obtainedfrom 20 CF patients during the first year of life fromFebruary 1999 to July 2007. The results of these culturesare shown in Table 2. The predominant organisms found inCF patients were S. aureus (28%) and Candida species

(28%), followed by E. cloacae (18.7%). Ps. aeruginosawas found in 14.7% of cultures. The 11 samples positivefor Ps. aeruginosa were obtained from nine patients andoccurred predominantly in the oldest age group (9 monthto 1 year). Smaller percentages of H. influenzae (5.3%)and M. catarrhalis (2.7%) were seen in the CF groupwhen compared to the non-CF group. Figure 1 showspercentages of subjects growing particular organisms, andcompares CF to non-CF subjects. Those patients withmilder mutations were as likely to have gram-negativeorganisms in their cultures as those with more severemutations.

DISCUSSION

Our results indicate that pathogens typically associatedwith pediatric CF disease such as S. aureus, H. influenzae,St. maltophilia, and Ps. aeruginosa can be recovered from

TABLE 1— Results of Oropharyngeal Cultures From Healthy, Non-CF Infants by Age Group

3 days–3 months

(n¼ 22)

3 months–6 months

(n¼ 21)

6 months–9 months

(n¼ 20)

9 months–1 year

(n¼ 20) % (n¼ 83)

S. aureus 5 6 7 5 27.7

Candida spp. 3 4 4 2 15.7

E. cloacae 2 5 2 0 10.8

E. coli 3 2 4 0 10.8

H. influ 1 1 5 2 10.8

M. catarrhalis 4 1 3 1 10.8

Klebsiella 3 1 2 2 9.6

Acinetobacter spp. 1 2 1 2 7.2

H. parainf 1 2 0 0 3.6

Ps. aeruginosa 0 1 0 2 3.6

St. maltophilia 1 1 1 0 3.6

Ps. putida 0 1 1 0 2.4

B. bronchoseptica 1 0 1 0 2.4

Many infants had several bacteria in their cultures. Percentages in the final column reflect results for infants over 48 hr of age (n¼ 83).

TABLE 2— Results of Oropharyngeal Cultures From CF Infants in the First Year of Life by Age Group

3 day–3 months

(n¼ 19)

3 months–6 months

(n¼ 19)

6 months–9 months

(n¼ 11)

9 months–1 year

(n¼ 26) % (n¼ 75)

Candida spp. 4 6 2 9 28.0

S. aureus 4 7 4 6 28.0

E. cloacae 3 4 3 4 18.6

P. aeruginosa 1 1 2 7 14.6

E. coli 2 1 2 4 12.0

Klebsiella 2 1 1 2 8.0

St. maltophilia 1 1 0 4 8.0

H. influenza 1 1 0 2 5.3

H. parainfluenza 0 1 0 3 5.3

Acinetobacter spp. 2 1 0 0 4.0

M. catarrhalis 1 1 0 0 2.7

B. bronchiseptica 0 0 1 0 1.3

P. putida 0 0 0 0 0

Many infants had several bacteria in their cultures.

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Oropharyngeal Flora in Healthy Infants 499

the oropharynx of non-CF affected infants. Discountingthe 21 infants cultured in the first 48 hr of life who had notyet had time to acquire anything more than the mostbasic maternal vaginal flora, we found that 27% (23/83) ofinfants had S. aureus in their oropharyngeal cultures,11% (9/83) H. influenzae, and 11% (9/83) E. coli. Three of83 cultures were positive for Ps. aeruginosa (3.6%), while2 others were positive for Ps. putida, an organismgenerally considered to be non-pathogenic.11 A consi-derable number of other gram-negative organisms werefound in normal infants’ oral cavities, consistent with theidea that there is transmission of maternal vaginal flora atbirth as well as fecal–oral contamination later in life.Many cultures grew multiple organisms.

The data from the CF patients is not strictly comparableto the well child data since the former consists oflongitudinal information collected on a small set ofpatients and the latter cross-sectional data on a largecohort. However, it is worth noting the presence of manyof the same organisms in the oropharynx of bothgroups in fairly similar proportions, but with more gram-negative organisms recovered from CF patients as theyage (Fig. 1).

Until recently there has been little need for physicians toknow what bacteria might be present in the normaloropharynx outside of the neonatal period. With theadvent of CF newborn screening, physicians are nowperforming routine cultures of oropharyngeal secretionsin very young infants. Thus, it is increasingly important tounderstand what ‘‘normal’’ is in order to be able to make

sense of the results of these diagnostic studies. Bacterialcolonization of the gastrointestinal tract and nasopharynxbegins with passage through the birth canal and evolvesover the first few weeks of life.12–14

Prior studies of infant oropharyngeal colonizationhave differed from ours and are not applicable tothe dilemma CF care providers face. In 1994, Finelliet al.9 obtained weekly oropharyngeal cultures from147 low birth weight infants in a neonatal intensivecare unit. Clearly, these babies are not comparable toolder, CF outpatients. Of note, however, S. aureus wasfound in 11.7% of cultures and Ps. aeruginosa was foundin 2.3%. As in our sample, polymicrobial cultureswere common. In another group of premature infants(24–34 week gestation), Makhoul et al.10 found that at10 days of age the most common oral bacteria ininfants not receiving antibiotics were alpha-hemolyticstreptococci, Klebsiella pneumoniae, and E. coli. Ps.aeruginosa was found in 2 of 23 infants who did notreceive antibiotic therapy and in another 3 of 39 infantswho did receive antibiotics. Bisgaard et al.5 investigatedchildren in the Copenhagen Prospective Study on Asthmain Childhood. Aspirates from the hypopharynx ofasymptomatic 1-month-old infants were cultured for S.pneumoniae, H. influenzae, M. catarrhalis, and S. aureus.Samples were obtained from 321 infants; 61% werecolonized with S. aureus, 9% with S. pneumoniae, 9%with H. influenzae, and 8% with M. catarrhalis.Unfortunately, these investigators did not look for gram-negative organisms such as E. coli, K. pneumoniae, or Ps.

Fig. 1. Percentage of cultures positive for specific bacteria in CF and non-CF individuals.

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500 Carlson et al.

aeruginosa, nor were cultures obtained at more than1 month of age.

We found that three healthy, non-CF infants had non-mucoid Ps. aeruginosa in their oropharynx (3.6%). Thispercentage of Ps. aeruginosa is similar to that reported byFinelli et al.9 and Makhoul et al.10 in newborns. None ofour infants had the mucoid variant of this organism, whichis commonly seen in chronically infected CF patients.Previous studies have shown the presence of pseudo-monads in fecal cultures8 and it is presumed that oursubjects had fecal–oral transmission of Ps. aeruginosa.Although not as common as S. aureus or E. coliin healthy infants, the presence of non-mucoid Ps.aeruginosa in an upper airway culture should not beconsidered pathognomonic for CF. Even though apositive oropharyngeal culture does not reliably rule-inthe presence of Ps. aeruginosa in the lower airways ofinfants with CF,15 given the deleterious consequences ofchronic infection with this organism and its abilityto transform into a mucoid producing strain that maybe impossible to eradicate, a child with a known CFgenotype who is Ps. aeruginosa-positive should be treatedaggressively.16,17

There are limitations to this preliminary study. This wasa survey of healthy infants who had negative newbornscreening for CF. We did not perform sweat tests orgenotyping on any subjects to confirm the absence of CFor determine CF carrier status. In addition, we did not lookfor anaerobic bacteria; however, since recommendationsfor routine CF culture do not include methods for isolationof anaerobic bacteria we do not think this impinges on therelevance of our study. All research subjects were felt to bewell by their primary care doctors, but it is possible thatrare occurrences of primary ciliary dyskinesia, immunedeficiencies, or neuromuscular disorders that could leadto chronic aspiration and/or changes in the normal upperairway flora were present in our cohort. Finally, althoughwe obtained environmental data on tobacco smoke,breast milk, and antibiotic exposure, our sample sizewas too small to determine if any of these play a role inoropharyngeal bacterial colonization. Recognizingenvironmental factors that could influence infectionand inflammation in infancy could lead to changes inmanagement for our youngest patients. A larger studycomparing healthy non-CF patients to their CF counter-parts might provide evidence that limiting or enhancingsome environmental exposures (e.g., tobacco smoke,breast-feeding) might impact oral flora and possibly affectlower respiratory tract colonization and subsequentinfection in CF patients.

This study is an important first step in determiningthe normal flora in healthy infants up to 1 year of age. Weused the same swabs that we use to obtain culturesspecimens from CF infants and processed them in thesame manner. The results reported here reflect the

common occurrence of S. aureus, H. influenzae, andseveral gram-negative bacteria in the oropharynx ofhealthy infants up to 1 year of age. The presence of theseorganisms in surveillance cultures from the oropharynx ofa child with a borderline sweat test and atypical CFmutation should not be construed as being confirmatory ofthe diagnosis of CF.

ACKNOWLEDGMENTS

We wish to thank the staff of the microbiologylaboratory for their hard work and assistance in thisproject. In addition, we thank Dr. Linda Sagor, Dr. LyndaYoung, and Dr. Mark Vining for allowing us access to theirpatients.

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