In Vitro Bacterial Interference in the Nasopharynx of Otitis Media–Prone and Non–Otitis Media–Prone Children

Objective  To compare the frequency of recovery of potential pathogens and aerobic- and anaerobic-interfering bacteria in the nasopharynx of otitis media–prone (OMP) with that in non-OMP (N-OMP) children.

Patients and Methods  Nasopharyngeal cultures were obtained from 20 OMP and 20 N-OMP children. Potential pathogens and aerobic and anaerobic bacteria with interfering capabilities against these organisms were identified.

Results  Eighteen potential pathogens were isolated from 12 of the 20 OMP children, and 9 were recovered from 5 of the 20 N-OMP children (P<.05). Fifty-eight aerobic and anaerobic isolates with interfering capability against 4 potential pathogens were recovered from 5 of the OMP group, and 139 from 17 of the N-OMP group (P<.05). These interfering organisms included αhemolytic streptococci, nonhemolytic streptococci, Prevotella species, and Peptostreptococcus species.

Conclusion  The nasopharyngeal flora of N-OMP children contains more aerobic and anaerobic organisms with interfering capability and less potential pathogens than that of OMP children.

THE NASOPHARYNX of normal children is generally colonized by relatively nonpathogenic aerobic and anaerobic organisms,¹ some of which possess the ability to interfere with the growth of potential pathogens.^2,3 The interfering organisms that have been the subject of most studies are aerobic αhemolytic streptococci (mostly Streptococcus mitis and Streptococcus sanguis).⁴ Conversely, carriage of potential respiratory pathogens, such as Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis, increases significantly in otitis media–prone (OMP) children and in the general population of young children during respiratory illness.^5-8 However, to our knowledge, there have been no previous studies exploring the role of interfering organisms other than aerobic organisms in otitis media.

This study was designed to compare the recovery of upper respiratory tract pathogens and all interfering aerobic and anaerobic bacteria from the nasopharyngeal flora of OMP and non-OMP (N-OMP) children.

Forty children who were seen consecutively by the authors in an outpatient clinic, 20 with OMP and 20 with N-OMP, were included in the study. The patients ranged in age from 18 to 52 months (average age, 35 months), and 29 were boys. Age and sex distribution were similar in both groups. The OMP children were defined, based on medical records, as patients who had had at least 6 episodes of acute suppurative otitis media in the previous 2 years, and N-OMP children were defined as those who had had fewer than 3 episodes in the previous 2 years.

Nasopharyngeal cultures, which were obtained at regular annual physical examination visits, were taken with calcium alginate swabs that were immediately plated into media supportive of the growth of aerobic and anaerobic bacteria. None of the children had undergone a prior adenoidectomy or tonsillectomy, had perforation of the tympanic membranes, had been treated with antimicrobials or other medications, or had suffered from any infections in the preceding 4 weeks, and none suffered from chronic medical problems.

Sheep's 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 under 5% carbon dioxide (blood and chocolate agars), and they were examined at 24 and 48 hours. For the recovery of anaerobic bacteria, the specimens were inoculated onto prereduced phytonadione-enriched Brucella blood agar, blood agar that contained kanamycin sulfate and vancomycin hydrochloride, and an aerobic blood plate that contained phenylethyl alcohol and enriched thioglycolate broth. These media were incubated in anaerobic jars at 37°C and examined after 48 and 96 hours of incubation at 37°C. Aerobic and anaerobic bacteria were identified by previously described methods.^9,10

The inhibitory activity was tested against 1 strain each of a recent clinical isolate of S pneumoniae, H influenzae, M catarrhalis, and Streptococcus pyogenes. The inhibitory activity of 5 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 phytonadione-enriched Brucella blood or chocolate (for H influenzae) agar plates and allowed to dry for 15 minutes at room temperature. A sample of a log-phase broth culture of the target strain was applied adjacently to each of the isolated strains, and the plates were incubated in 5% carbon dioxide or anaerobically at 37°C for 48 hours. Bacterial interference was defined as any reproducible inhibition of growth. The 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 χ² test with Yates correction.

Eighteen potential pathogens were isolated from 12 of the 20 OMP children, and 9 were recovered from 5 of the 20 N-OMP children (P<.05) (Table 1).

Fifty-eight aerobic and anaerobic isolates with interfering capability against 4 potential pathogens were recovered from 5 of the OMP group, and 139 from 17 of the N-OMP group (P<.05 ). The isolates included aerobic (α-hemolytic and nonhemolytic streptococci) and anaerobic (Prevotella and Peptostreptococcus species) organisms (Table 2). No correlation was found between the microbiological findings and age or sex of the children.

Our study compared the recovery rate of potential pathogens as well as interfering aerobic and anaerobic bacteria from OMP and N-OMP children. As was previously described,^4-8H influenzae, S pneumoniae, and M catarrhalis were more frequently isolated in OMP than in N-OMP patients. We were also able to demonstrate, as was reported by Bernstein et al⁴ and Fujimori et al,⁸ a higher recovery of interfering α-hemolytic streptococci in N-OMP children than in those who are OMP. The presence of organisms with interfering potential may play a role in the prevention of otitis media. Alternatively, it is possible that the previous frequent use of antibiotics in children who are OMP may have reduced the number of organisms that are inhibitory to the growth of pathogens. It is yet to be determined if using antibiotics with wide-spectrum efficacy against members of the oral flora may enhance colonization with potential pathogens.

We were able to show for the first time that organisms other than α-hemolytic streptococci can also interfere with the in vitro growth of potential respiratory tract pathogens. These organisms include aerobic nonhemolytic streptococci as well as the anaerobic bacteria Peptostreptococcus and Prevotella species. The potential in vitro inhibitory activity of these 2 genera of anaerobic bacteria was previously reported.¹²

The ability of the indigenous normal nasopharyngeal flora to inhibit colonization with potential pathogens has been studied. α-Hemolytic streptococci were found to inhibit the colonization and in vitro growth of a variety of pathogenic bacteria, including S pneumoniae, S pyogenes, and Staphylococcus aureus.^13-17 The production of bacteriocins and other inhibitory substances that suppress some bacterial growth, or the use of nutrients in the nasopharyngeal environment that are essential for the growth of potential pathogens, may explain this phenomenon.¹⁸

Therapeutic colonization of the nasopharynx with interfering bacteria was studied by Roos et al,¹⁹ who implanted children with repeated tonsillitis with either αhemolytic streptococci or placebo. Clinical recurrences occurred in 1 (2%) of the 51 children in the α-hemolytic streptococci group and in 14 (23%) of the 61 children in the placebo-treated group. It is possible that maintenance of the normal nasopharyngeal flora that possesses inhibitory potential of pathogens can contribute to the reduction of recurrent ear infection. Further studies are warranted to investigate whether maintaining normal flora in the nasopharynx would be beneficial in OMP children.

Accepted for publication March 13, 2000.

The authors acknowledge the laboratory support of James Perry and Thom Elliott, PhD, and the secretarial support of Joanie Pietrafitta.

Corresponding author: Itzhak Brook, MD, MSc, PO Box 70412, Chevy Chase, MD 20813-0412.